Taxonomic status and remarks on ecology of the Malawian mole-ratCryptomys whytei (Rodentia, Bathyergidae)

Mole-rats from Karonga, Malawi, the type locality ofCryptomys hottentotus whytei, and a single female mole-rat from the Malawian Nyika Plateau exhibit a karyotype 2n = 4 6 chromosomes, wit h 1 5 metacentr ic an d 8 acrocentr ic chromosome, s ie NF (female) = 76. This trait, together with several morphological features and a previously published genetic analysis (Ingramet al. 2004), identifies this species as a good species, unrelated toCryptomys hottentotus of South Africa, which belongs to the Zambezian clade of the genusCryptomys sensu lato (cf Ingramet al. 2004) and should be therefore called Whyte’s mole-rat,Cryptomys whytei. Available data on ecology and reproductive biology sugges t simila r characteristic s a s i n othe r mole-rat s o f th e genu.s Unexpecte d findin g of lar ge complicat ed undergroun d burro w system s contrasted, howev,e r wi th ve ry f ew abo ve grou nd moun.d s     

Transformation of folate-consuming Lactobacillus gasseri into a folate producer

Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri. As a result L. gasseri changed from a folate consumer to a folate producer. This principle can be used to increase folate levels in many fermented food products.     

Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1

Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappé et al., Biochim. Biophys. Acta 1520, 1-6, 2001). Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues. Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR. Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining. We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens. To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins. TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9. Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells. The possible functional significance of this interaction is discussed.     

Two new species of Araceae tribe Spathicarpeae from Bolivia

The tribe Spathicarpeae is one of the most poorly known-tribes in the Araceae. Recently, field collections in some areas of Bolivia have revealed many interesting members of this tribe. Two new species,Gorgonidium striatum Hett., P. L. Ibisch &; E. G. Gonç. andSpathantheum fallax Hett., P. L. Ibisch &; E. G. Gonç. are here described, illustrated, and compared to their closest relatives.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.     

Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.     

Increased production of folate by metabolic engineering of Lactococcus lactis

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.     

Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild-type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host.     

False-negative myocardial scintigraphy in balanced three-vessel disease,revealed by coronary pressure measurement

In nuclear perfusion imaging of the myocardium, a false-negative test result in patients with balanced three-vessel disease is a well-known pitfall. This paper describes a patient with typical chest pain and a negative myocardial perfusion scintigram. At coronary angiography, intermediate stenoses in the left anterior descending (LAD), left circumflex (LCX), and right coronary (RCA) arteries were present. Fractional flow reserve, measured by coronary pressure measurement, was 0.54, 0.56, and 0.66 respectively for the LAD, LCX, and RCA, unequivocally demonstrating the presence of balanced three-vessel disease. The patient underwent successful bypass surgery and remained event-free thereafter.     

Selective DNA damage responses in murine Xpa-/-, Xpc-/- and Csb-/- keratinocyte cultures

Cellular DNA damage responses (DDRs) are induced by unrepaired DNA lesions and constitute a protective back-up system that prevents the expansion of damaged cells. These cellular signaling pathways trigger either growth arrest or cell death and are believed to be major components of an early anti-cancer barrier. Cultures of C57BL/6J keratinocytes with various defects in NER sub-pathways allowed us to follow the kinetics of DDRs in an isogenic background and in the proper (physiologically relevant) target cells, supplementing earlier studies in heterogenic human fibroblasts. In a series of well-controlled parallel experiments we have shown that, depending on the NER deficiency, murine keratinocytes elicited highly selective DDRs. After a dose of UV-B that did not affect wild-type keratinocytes, Xpa(-/-) keratinocytes (complete NER deficiency) showed a rapid depletion of DNA replicating S-phase cells, a transient increase in quiescent S-phase cells (not replicating DNA), followed by massive apoptosis. Csb(-/-) keratinocytes (TC-NER deficient) responded by a more sustained increase in QS-phase cells and appeared more resistant to UV-B induced apoptosis than Xpa(-/-). In irradiated Xpc(-/-) keratinocytes (GG-NER deficient) the loss of replicating S-phase cells was associated with a gradual build-up of both QS-phase cells and cells arrested in late-S phase, in complete absence of apoptosis. Our analysis complements and extends previous in vivo investigations and highlights both similarities and differences with earlier fibroblast studies. In vitro cultures of murine keratinocytes provide a new tool to unravel the molecular mechanisms of UV-induced cellular stress responses in great detail and in a physiologically relevant background. This will be essential to fully appreciate the implications of DDRs in tumor suppression and cancer prevention.