Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)Glc-Nac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

Development and characterisation of recombinant hepatitis delta virus-like particles

Injection of particulate hepatitis B virus surface antigen (HBsAg) in mice leads to the induction of a HBsAg-specific class-I-restricted cytotoxic T lymphocyte (CTL) response. It is proposed that any protein internal to HBsAg will also be able to elicit a specific CTL response. In this study, several carboxy-terminal truncations of hepatitis C virus (HCV) core protein were fused to varying lengths of amino-terminal truncated large hepatitis delta antigen (L-HDAg). These constructs were analysed for their ability to be expressed and the particles secreted in the presence of HBsAg after transfection into HuH-7 cells. The secretion efficiency of the various HCV core-HDAg chimeric proteins was generally poor. Constructs containing full length HDAg appeared to be more stable than truncated versions and the length of the inserted protein was restricted to around 40 amino acids. Thus, the use of L-HDAg as a chimera to package foreign proteins is limited. Consequently, a polyepitope (polytope) containing a B-cell epitope from human papillomavirus (HPV 16) and multiple T-cell epitopes from the HCV polyprotein was used to create the construct, L-HDAg-polyB. This chimeric protein was shown to be reliant on the co-expression of HBsAg for secretion into the cell culture fluid and was secreted more efficiently than the previous HCV core-HDAg constructs. These L-HDAg-polyB virus-like particles (VLPs) had a buoyant density of similar to 1.2 g/cm(3) in caesium chloride and similar to 1.15 g/cm(3) in sucrose. The VLPs were also immunoprecipitated using an anti-HBs but not an anti-HD antibody. Thus, these recombinant VLPs have similar biophysical properties to L-HDAg VLPs.

Adult mouse intrinsic laryngeal muscles express high levels of the myogenic regulatory factor, MYF-5

The intrinsic laryngeal muscles display unique structural and functional characteristics that distinguish them from the skeletal muscle of the trunk and limbs. These features include relatively small muscle fibers, super-fast contraction speed, and fatigue resistance. The molecular basis of tissue-specific functions and other characteristics is differential gene expression. Accordingly, we have investigated the molecular basis of the functional specialization of the intrinsic laryngeal muscles by examining the expression of two key genes in the larynx, known to be important for skeletal muscle development and function: (a) the muscle regulatory factor, Myf-5, and (b) the superfast-contracting myosin heavy chain (EO-MyHC). We have found that the adult thyroarytenoid muscles express much higher levels of both Myf-5 and EO-MyHC messenger ribonucleic acid (mRNA), compared to lower hindlimb skeletal muscle where Myf-5 mRNA levels are very low and EO-MyHC is not detectable. These findings suggest that the unique functional characteristics of the intrinsic laryngeal muscles may be based in laryngeal muscle-specific gene expression directed by a unique combination of muscle regulatory factors. Such laryngeal muscle-specific genes may allow the future development of new treatments for laryngeal muscle dysfunction.

Maturation studies of pecan nuts grown in Queensland

Changes in chemical composition, physical and sensory characteristics were followed in two pecan cultivars Wichita and Western Schley harvested from a commercial orchard at Gatton in Queensland seven times during 1996. Testa colour of both pecan cultivars darkened and opalescence decreased as the nuts matured. Bitterness of Western Schley pecans decreased with maturity. Colour of shuck, shell and kernel of both cultivars developed as the nuts matured. Wichita pecans were larger than Western Schley at all harvest times. Both nut-in-shell and kernel moisture decreased with maturity, whereas oil and sucrose contents increased. Both pecan cultivars had reached advanced maturation by the first harvest on March 18.

Composition of pecan cultivars Wichita and Western Schley [Carya illinoinensis (Wangenh.) K. Koh] grown in Australia

Pecans from the cultivars Wichita and Western Schley [Carya illinoinensis (Wangenh.) K. Koch] collected over three years were analyzed for the following constituents: total lipid content; fatty acid profiles; sucrose content; protein; total dietary fiber; the minerals magnesium, calcium, potassium, sulfur, phosphorus, boron, copper, iron, manganese, sodium, zinc, and aluminum; vitamin C; and lipase; and lipoxygenase activities. Year of harvest and cultivar had little effect on the composition of the pecans. Overall, protein content was the only constituent that differed between pecans grown in Australia and those grown in the United States. This difference is probably related to differences in growing location and horticultural practices between the two countries.

Eutectic solidification mode in sodium modified Al-7 mass %Si-3.5 mass %Cu-0.2 mass %Mg casting alloys

The influence of sodium (Na) on nucleation and growth of the Al-Si eutectic in a commercial hypoeutectic Al-Si-Cu-Mg foundry alloy has been investigated. The microstructural evolution during eutectic solidification was studied by a quenching technique. By comparing the orientation of the aluminium in the eutectic to that of the surrounding primary aluminium dendrites by EBSD, the eutectic solidification mode could be determined. The results show that the eutectic solidification starts near the mould wall and evolves with front growth opposite the thermal gradient on a macro-scale, and on a micro-scale with independent heterogeneous nucleation of eutectic grains in interdendritic spaces. Na-modified alloys therefore behave significantly differently from those modified by other elemental additions.

Assessing ecological impacts of shrimp and sewage effluent: Biological indicators with standard water quality analyses

Despite evidence linking shrimp farming to several cases of environmental degradation, there remains a lack of ecologically meaningful information about the impacts of effluent on receiving waters. The aim of this study was to determine the biological impact of shrimp farm effluent, and to compare and distinguish its impacts from treated sewage effluent. Analyses included standard water quality/sediment parameters, as well as biological indicators including tissue nitrogen (N) content, stable isotope ratio of nitrogen (delta N-15) and amino acid composition of inhabitant seagrasses, mangroves and macroalgae. The study area consisted of two tidal creeks, one receiving effluent from a sewage treatment plant and the other from an intensive shrimp farm. The creeks discharged into the western side of Moreton Bay, a sub-tropical coastal embayment on the east coast of Australia. Characterization of water quality revealed significant differences between the creeks, and with unimpacted eastern Moreton Bay. The sewage creek had higher concentrations of dissolved nutrients (predominantly NO3-/NO2- and PO43-, compared to NH4+ in the shrimp creek). In contrast, the shrimp creek was more turbid and had higher phytoplankton productivity. Beyond 750 m from the creek mouths, water quality parameters were indistinguishable from eastern Moreton Bay values. Biological indicators detected significant impacts up to 4 km beyond the creek mouths (reference site). Elevated plant delta N-15 values ranged from 10.4-19.6 parts per thousand at the site of sewage discharge to 2.9-4.5 parts per thousand at the reference site. The free amino acid concentration and composition of seagrass and macroalgae was used to distinguish between the uptake of sewage and shrimp derived N. Proline (seagrass) and serine (macroalgae) were high in sewage impacted plants and glutamine (seagrass) and alanine (macroalgae) were high in plants impacted by shrimp effluent. The delta N-15 isotopic signatures and free amino acid composition of inhabitant flora indicated that sewage N extended further from the creek mouths than shrimp N. The combination of physical/chemical and biological indicators used in this study was effective in distinguishing the composition and subsequent impacts of aquaculture and sewage effluent on the receiving waters. (C) 2001 Academic Press.

Effects of concentrated viral communities on photosynthesis and community composition of co-occurring benthic microalgae and phytoplankton

Marine viruses have been shown to affect phytoplankton productivity; however, there are no reports on the effect of viruses on benthic microalgae (microphytobenthos). Hence, this study investigated the effects of elevated concentrations of virus-like particles on the photosynthetic physiology and community composition of benthic microalgae and phytoplankton. Virus populations were collected near the sediment surface and concentrated by tangential flow ultrafiltration, and the concentrate was added to benthic and water column samples that were obtained along a eutrophication gradient in the Brisbane River/Moreton Bay estuary, Australia. Photosynthetic and community responses of benthic microalgae, phytoplankton and bacteria were monitored over 7 d in aquaria and in situ. Benthic microalgal communities responded to viral enrichment in both eutrophic and oligotrophic sediments. In eutrophic sediments, Euglenophytes (Euglena sp.) and bacteria decreased in abundance by 20 to 60 and 26 to 66%, respectively, from seawater controls. In oligotrophic sediments, bacteria decreased in abundance by 30 to 42% from seawater controls but the dinoflagellate Gymnodinium sp. increased in abundance by 270 to 3600% from seawater controls, The increased abundance of Gymnodinium sp. may be related to increased availability of dissolved organic matter released from lysed bacteria. Increased (140 to 190% from seawater controls) initial chlorophyll a fluorescence measured with a pulse-amplitude modulated fluorometer was observed in eutrophic benthic microalgal incubations following virus enrichment, consistent with photosystem II damage. Virus enrichment in oligotrophic water significantly stimulated carbon fixation rates, perhaps due to increased nutrient availability by bacterial lysis. The interpretation of data from virus amendment experiments is difficult due to potential interaction with unidentified bioactive compounds within seawater concentrates. However, these results show that viruses are capable of influencing microbial dynamics in sediments.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

A high efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

A novel target-field method for finite-length magnetic resonance shim coils: I. Zonal shims

This paper presents a new approach for the design of genuinely finite-length shim and gradient coils, intended for use in magnetic resonance imaging equipment. A cylindrical target region is located asymmetrically, at an arbitrary position within a coil of finite length. A desired target field is specified on the surface of that region, and a method is given that enables winding patterns on the surface of the coil to be designed, to produce the desired field at the inner target region. The method uses a minimization technique combined with regularization, to find the current density on the surface of the coil. The method is illustrated for linear, quadratic and cubic magnetic target fields located asymmetrically within a finite-length coil.

Asymmetric zonal shim coils for magnetic resonance applications

A method is presented for the systematic design of asymmetric zonal shim coils for magnetic resonance applications. Fourier-series methods are used to represent the magnetic field inside and outside a circular cylinder of length 2L and radius a. The current density on the cylinder is also represented using Fourier series. Any desired field can be specified in advance on the cylinder's radius, over some nonsymmetric portion pL

Compact clinical MRI magnet design using a multilayer current density approach

In this work, a new method of optimization is successfully applied to the theoretical design of compact, actively shielded, clinical MRI magnets. The problem is formulated as a two-step process in which the desired current densities on multiple, cc-axial surface layers are first calculated by solving Fredholm equations of the first kind. Non-linear optimization methods with inequality constraints are then invoked to fit practical magnet coils to the desired current densities. The current density approach allows rapid prototyping of unusual magnet designs. The emphasis of this work is on the optimal design of short, actively-shielded MRI magnets for whole-body imaging. Details of the hybrid numerical model are presented, and the model is used to investigate compact, symmetric, and asymmetric MRI magnets. Magnet designs are presented for actively-shielded, symmetric magnets of coil length 1.0 m, which is considerably shorter than currently available designs of comparable dsv size. Novel, actively-shielded, asymmetric magnet designs are also presented in which the beginning of a 50-cm dsv is positioned just 11 cm from the end of the coil structure, allowing much improved access to the patient and reduced patient claustrophobia. Magn Reson Med 45:331540, 2001. (C) 2001 Wiley-Liss, Inc.