Fast pollen tube growth in Conospermum species.

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.

Maintenance of rat taste buds in primary culture.

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.

Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction.

BACKGROUND: Taste receptor cells are responsible for transducing chemical stimuli into electrical signals that lead to the sense of taste. An important second messenger in taste transduction is IP3, which is involved in both bitter and sweet transduction pathways. Several components of the bitter transduction pathway have been identified, including the T2R/TRB taste receptors, phospholipase C beta2, and the G protein subunits alpha-gustducin, beta3, and gamma13. However, the identity of the IP3 receptor subtype in this pathway is not known. In the present study we used immunocytochemistry on rodent taste tissue to identify the IP3 receptors expressed in taste cells and to examine taste bud expression patterns for IP3R3. RESULTS: Antibodies against Type I, II, and III IP3 receptors were tested on sections of rat and mouse circumvallate papillae. Robust cytoplasmic labeling for the Type III IP3 receptor (IP3R3) was found in a large subset of taste cells in both species. In contrast, little or no immunoreactivity was seen with antibodies against the Type I or Type II IP3 receptors. To investigate the potential role of IP3R3 in bitter taste transduction, we used double-label immunocytochemistry to determine whether IP3R3 is expressed in the same subset of cells expressing other bitter signaling components. IP3R3 immunoreactive taste cells were also immunoreactive for PLCbeta2 and gamma13. Alpha-gustducin immunoreactivity was present in a subset of IP3R3, PLCbeta2, and gamma13 positive cells. CONCLUSIONS: IP3R3 is the dominant form of the IP3 receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

A variation at position -30 of the beta-cell glucokinase gene promoter is associated with reduced beta-cell function in middle-aged Japanese-American men.

We sought to determine whether a G -> A variant at position -30 of the beta-cell promoter of the glucokinase (GCK) gene observed to be present more frequently in Japanese-American men with impaired glucose tolerance (IGT) than in Japanese-American men with normal glucose tolerance (NGT) is associated with impaired beta-cell function. We studied 125 unrelated Japanese-American men (aged 46-74 years; mean 61 +/- 0.5) who were nondiabetic by a 75-g oral glucose tolerance test (OGTT) (65 had NGT and 60 had IGT). The presence of the -30 beta-cell GCK gene promoter variant was determined by single-strand conformation polymorphism analysis. Beta-cell function was assessed using the ratio of the incremental response in immunoreactive insulin (IRI) to that of glucose during the first 30 min of the OGTT (delta IRI[30 min-0 min]/delta glucose[30 min-0 min]) performed at baseline and at 5 years of follow-up. Beta-cell function adjusted for basal IRI ([delta IRI[30 min-0 min]/delta glucose[30 min-0 min]]/basal IRI; the relative insulin response) was also evaluated. At baseline, the -30 beta-cell GCK gene promoter variant was present in 15.4% of subjects with NGT vs. 38.3% of subjects with IGT (P < 0.01). Fasting IRI did not differ between groups. At baseline, delta IRI[30 min-0 min]/delta glucose[30 min-0 min] was significantly lower in subjects with the promoter variant (57 x 10(-9) [35 x 10(-9) to 95 x 10(-9)] vs. 77 x 10(-9) [55 x 10(-9) to 128 x 10(-9)]; median [interquartile range]; P < 0.01) as was the relative insulin response (0.97 [0.70-1.24] vs. 1.37 [0.95-2.03]l/mmol; P < 0.0005). Similarly, at 5 years of follow-up, delta IRI[30 min-0 min]/delta glucose[30 min-0 min] and the relative insulin response were significantly reduced in the group with the variant. In the subgroups of subjects with IGT at baseline, IGT at 5 years, and NGT at 5 years, the relative insulin response was significantly lower in those with the variant. We conclude that the -30 beta-cell GCK gene promoter variant is associated with reduced beta-cell function in middle-aged Japanese-American men and may contribute to the high risk of abnormal glucose tolerance in this population.

Taste receptor cells arise from local epithelium, not neurogenic ectoderm.

Except for taste bud cells, all sensory receptor cells and neurons have been shown to originate from neurogenic ectoderm (i.e., neural tube, neural crest, or ectodermal placodes). Descriptive studies on taste buds indicate that they, however, may arise from local epithelium. To determine whether taste receptor cells originate from neurogenic ectoderm or from local epithelium, the tongues of X chromosome-inactivation mosaic mice were examined. Results of this analysis show that taste bud cells and their surrounding epithelium always match in terms of the mosaic marker. This suggests that taste cells and epithelial cells arise from a common progenitor and that taste receptor cells originate from local tissue elements. Since taste buds are widespread in the oropharynx, they lie in epithelium derived from both ectoderm and endoderm. Therefore, taste receptor cells can be induced in tissue from two different germ layers. Thus in terms of tissues of origin, taste receptor cells are unlike other cells with neuronal characteristics.

Mosaic analysis of the embryonic origin of taste buds.

The embryonic origins of taste receptor cells have not been established experimentally. Although related receptor cells (e.g. hair cells of the inner ear, lateral line receptors) are known to arise from neurogenic ectoderm (e.g. neural crest or placodes), taste buds are described as arising from local epithelial cells. Also unknown is whether or not each taste bud is a clone of cells, i.e. arising from a single progenitor. To address these problems, mosaic and chimeric analyses of lingual epithelium and taste buds have been undertaken. This paper describes the theory of chimeric and mosaic cell lineage analyses, the advantages and disadvantages, and the preliminary results obtained from the examination of the taste buds and lingual epithelium of: 1) mosaic Xenopus, 2) chimeric mice and 3) X-inactivation mosaic mice.

Glucokinase gene variations in Japanese-Americans with a family history of NIDDM.

OBJECTIVE: To determine if sequence variants in the glucokinase (GCK) gene contribute to the high risk of impaired glucose metabolism in Japanese-Americans and whether the gene sequence differs between Japanese-Americans and Caucasians. RESEARCH DESIGN AND METHODS: Forty-seven unrelated Japanese-Americans with one or more first-degree relatives with non-insulin-dependent diabetes mellitus (NIDDM) were selected, irrespective of glucose tolerance status. By World Health Organization criteria, 13 had normal glucose tolerance, 11 had impaired glucose tolerance, and 23 had NIDDM. Variations in the GCK gene were identified by single-strand conformation polymorphism analysis and sequenced using standard techniques. RESULTS: Six variants of the GCK gene were identified in a total of 21 subjects: 1) a G--> A substitution at nucleotide -30 in the beta-cell-specific promoter; 2) an A--> G substitution at nucleotide 244 in the 5'-untranslated region (5'-UTR) of exon 1a; 3) a C--> G substitution at nucleotide 403 in the 5'-UTR of exon 1a; 4) a G--> A variant 13 base pair (bp) 5' to the intron 3 exon 4 junction; 5) a silent substitution in the third base of codon 145 in exon 4; and 6) a C--> T substitution 8 bp 3' to the exon 9 intron 9 junction. None of these variations would be expected to affect the structure of the GCK enzyme. While none of these variants were significantly associated with IGT or NIDDM, a nonsignificant increase in the beta-cell promoter variant was observed in subjects with abnormal glucose tolerance. No uniform sequence differences in the GCK gene were identified between Japanese-American and Caucasian-American subjects. CONCLUSIONS: Mutations affecting the amino acid sequence of GCK do not account for the increased incidence of impaired glucose metabolism in Japanese-Americans, and the gene sequence does not uniformly differ from that in Caucasians.

Differential distribution of the synapsins in the rat olfactory bulb.

The distribution of the different forms of synapsin in the rat olfactory bulb was investigated by biochemical and immunocytochemical methods. Western blots of tissue derived from microdissection of the surface and core regions of the olfactory bulb were performed using antibodies to synapsin I and synapsin II. The relative levels of the synapsins in the core region of the olfactory bulb were similar to the cerebral cortex. In contrast, the surface region of the olfactory bulb had significantly higher levels of synapsin IIa and significantly lower levels of synapsin I, relative to the cortex. Immunocytochemical localization of synapsin I and synapsin II revealed that synapsin I immunoreactivity was the most dense in the external plexiform layer and in the glomeruli; immunoreactivity was also present in the granule cell layer and the periglomerular regions. Synapsin II immunoreactivity was the most dense in the glomeruli. The external plexiform layer, internal plexiform layer, and granule cell layer exhibited much lower immunoreactivity. To determine the source of synapsin II immunoreactivity in the glomeruli, the olfactory epithelium was damaged to decrease the primary afferent input to the bulb. Three to four days later, olfactory bulb sections were double labeled with anti-olfactory marker protein (OMP) antibodies and anti-synapsin II antibodies. Following denervation, both OMP and synapsin II immunoreactivities were diminished, and continued to colocalize in regions retaining immunoreactivity. Individual puncta were immunoreactive for both OMP and synapsin II. Occasional puncta contained only synapsin II immunoreactivity. These results indicate that the distribution of the synapsins in the olfactory bulb differs from most other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)