Optimal conditions for breaking medically important yeasts by an inexpensive and simple method

Conditions for breaking various medically important yeasts using glass beads, 30 ml Corex centrifuge tubes, and a Vortex mixer were determined. From 75–95% ofCandida hyphal cells and all species of yeasts exceptSporothrix schenckii were broken when 10 g of 0.45–0.50 mm glass beads, 50–300 mg of wet cells in 5 ml of buffer, and 90 s of vortexing were employed. Yeasts ofSporothrix schenckii broke more efficiently when 0.25–0.30 mm beads were used.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Clonal populations of the mouse mammary cell line,COMMA-D,which retain capability of morphogenesis in vivo

Summary Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland. Supported by NCI research grants CA-38650, CA-33369, CA-39017, and CA-25215.     

Luteal phase defects: a possible relationship between short hyperthermic phase and sporadic sexual behavior in women

Sporadic (less than regular weekly) coital activity is associated with a particular type of infertility—the short hyperthermic (luteal) phase. This preliminary report is the first suggestion that a reduced frequency of human sexual activity might, itself, be associated with subfertile reproductive function.     

Differentiation of the myoepithelial cells of the rat submandibular gland in vivo and in vitro: an ultrastructural study

Cytodifferentiation of the myoepithelial cells (MEC) of the rat submandibular gland (SMG) was observed by studying the prenatal and postnatal development of the gland in vivo and in vitro by light and electron microscopy. The anlage of the SMG first appeared on the fourteenth day of gestation and, from its earliest inception, was surrounded by an intact basal lamina. Presumptive myoepithelial cells were first seen at 18 days of gestation coinciding with the onset of secretion in the rudiment. These cells were flattened, peripherally located and subjacent to the epithelial basal lamina. Initial deposition of cytofilaments in the MEC's was observed during the first three days following birth and fully matured cells were seen as early as one week after birth. Presumptive and immature MEC's were observed undergoing mitosis, but once cytofilament deposition had begun in the cells they did not divide. Myoepithelium developed in relation to embryonic secretory structures and were only observed surounding acini and intercalated ducts in the adult gland. New myoepithelial cells were formed as long as new acinar-intercalated duct units were formed. Myoepithelial cells did not produce secretory type granules at any time during their development or in their mature state. Development of the MEC's in vitro paralleled that in vivo and supported the above observations.