Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth

Vacuolar sorting receptors (VSRs) are type‐I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type‐IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans‐Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP‐VSR/GFP‐LIVSR is located throughout the pollen tubes, excepting the apical clear‐zone region, whereas GFP‐LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.     

Antibodies specific to isoniazid and isonicotinic acid.

Rabbit antisera to isoniazid (INH) and its major metabolite, isonicotinic acid (INA), were prepared by immunization with conjugates of these compounds with human serum albumin. The antisera were rendered hapten-specific by exhaustive absorption with the immunizing carrier. Purified anti-hapten antibodies were also isolated with appropriate immunosorbents. As demonstrated by inhibition of the quantitative precipitin curves and of precipitating immune complexes in immunodiffusion tests, the antibodies to the two haptens reacted with either INH or INA, and also with isonicotinamide (INC); these three related molecules share the isonicotinyl group. The relative effectiveness of inhibition by free hapten of precipitating immune complexes consisting of either anti-INH or anti-INA antibodies and the related hapten-protein conjugates was INH greater than INC greater than INA.     

Transitional cell carcinoma of the renal pelvis metastatic to the metacarpal. A case report correlating cytologic and histologic findings.

This report describes the cytologic, radiologic and histologic findings in a 76-year-old male who presented with a pathologic fracture of the first metacarpal bone as the result of metastatic transitional cell carcinoma. The primary neoplasm was sited in the right renal pelvis. Metastases were also detected in the liver and confirmed cytologically. Problems encountered with the cytologic diagnosis are explained by correlation with the histologic findings. The case also illustrates the importance of clinicopathologic correlation when interpreting fine needle aspiration biopsies.     

Quantitative determination of BAF312, a S1P-R modulator,in human urine by LC–MS/MS: Prevention and recovery of lost analyte due to container surface adsorption

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC–MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the ‘non-specific loss’ of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC–MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the ‘non-specific binding’ of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. β-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of β-cyclodextrin to untreated urine samples does not recover the ‘lost’ analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200–20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from ?3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid–liquid extraction in combination with an isocratic LC separation was employed. The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at ≤?60 °C and for at least 3 months when stored at ≤?60 °C. The current work could serve as a simple example in trouble shooting non-specific binding or container surface adsorption in quantitative analysis of urine samples.     

Chicken T lymphocyte clones with specificity for Eimeria tenella. I. Generation and functional characterization

T lymphocyte clones reacting specifically with the antigenic components of Eimeria tenella were generated from splenic lymphocytes of immunized chickens and were maintained for 12 to 14 wk in vitro. These T cell growth factor-dependent T lymphocyte clones from bursectomized and normal chickens proliferated in vitro when stimulated with antigens from different developmental stages of homologous but not heterologous species of the parasite. Specific proliferative responses of the cloned T cells showed an absolute requirement for antigen presentation by histocompatible antigen-presenting cells. Some of the T cell clones exhibited functionally discrete interactions with syngeneic primed B cells; 25% of the T cell clones from immunized normal chickens and 7% of those obtained from immunized bursectomized chickens showed antigen-dependent helper activity and induced specific antibody production by syngeneic primed B cells. Of the T cell clones from immunized normal chickens, 19% showed suppression of in vitro antibody production in comparison to 7% of those isolated from immunized bursectomized chickens. The frequency of cloned T cells with ability to induce cytotoxic activity in macrophages against the sporozoites of E. tenella was much higher in those isolated from bursectomized chickens (80%) than in those isolated from normal chickens. Because both bursectomized and normal chickens can be immunized by repeated infections, differences in the distribution among cloned T cells suggest different effector mechanisms of immunity against coccidiosis in these chickens. Lack of B cells seem to affect the development of T cell immunity as reflected by slower development of immunity and enhanced activation of cytotoxic T cell function.     

Solubilization and several properties of NAD-glucohydrolase isolated from rabbit heart]

Methods of isolation of NAD-glucohydrolase from the fraction of heart microsomes were searched for. NAD-glucohydrolase proved to pass into a soluble state under the effect of phospholipase A, triton X-100 and Na cholate. NAD-glucohydrolase was found to possess peculiar properties; it reversibly became denatured under the effect of 6M urea, but failed to become inactivated with own substrate (NAD) at pH 8.0. In case of NAD-ases isolated from other sources the reversible denaturation by means of urea as a rule correlated with the enzyme inactivation in the presence of NAD at pH 8.0.     

Complete genome sequences of novel canine noroviruses in Hong Kong

We report the complete genome sequences of two novel isolates of norovirus isolated from the fecal swab specimens of dogs in Hong Kong. The complete viral genome is approximately 7.6 kb in length and consists of 3 overlapping open reading frames encoding the ORF1 polyprotein, VP1, and VP2, respectively. Analysis of the VP1 sequence suggested that these noroviruses are divergent from known noroviruses and may represent a novel phylogenetic clade within the genus.