Multicompartmental distribution of the tuberous sclerosis gene products,hamartin and tuberin

Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.     

Multiplexed on-column protein digestion and capillary electrophoresis for high-throughput comprehensive peptide mapping

A novel scheme based on multiplexed capillary electrophoresis (CE) has been developed for high-throughput, low-cost and comprehensive peptide mapping. Orthogonal peptide maps of the protein of interest were obtained by using multiple reaction conditions with three different enzymes (trypsin, pepsin, and chymotrypsin), and multiple separation conditions with six zone electrophoresis buffers and two micellar electrokinetic chromatography (MEKC) buffers. Fifteen nanoliters of two protein samples (beta-lactoglobulin A and beta-lactoglobulin B) were separately mixed on-column and digested independently at 37 degrees C for 10 min to produce peptides in a 20-capillary system. The resulting peptides were detected simultaneously at 214 nm by a photodiode array detector. The overall analysis time from reaction to detection was about 40 min.     

Sperm volume regulation: maturational changes in fertile and infertile transgenic mice and association with kinematics and tail angulation

Laser light scatter analyzed by flow cytometry was used to monitor the volume of viable maturing murine spermatozoa. Upon release, dispersion, and dilution, epididymal sperm from fertile heterozygous c-ros knockout mice were smallest in the cauda region and largest in the corpus region. Cauda sperm from both infertile homozygous c-ros knockout and GPX5-Tag2 transgenic mice were abnormally large. When incubated, corpus and cauda sperm from normal mice became slightly enlarged and later returned to a smaller size. This suggests an immediate swelling due to high intracellular osmolality, which triggers a regulatory volume decrease (RVD) that results in a net volume reduction. Normal caput sperm increased in size continuously and became larger than the more mature sperm, indicating a lack of RVD. The ion-channel blocker quinine induced dose-dependent size increases in normal cauda sperm but not in caput sperm. Dose-dependent quinine action on mature sperm also included induction of tail angulation, and suppression of straight-line velocity and linearity. The kinematic effects were more sensitive, with a quicker onset, but they diminished with time in contrast to tail angulation, which intensified. These results suggest that kinematic changes are an early phenomenon of swelling, which gradually accumulates at the cytoplasmic droplet to cause flagellar angulation. Disruption of the epididymal maturation of sperm volume regulation capacity would hinder the transport of sperm in the female tract, and may thereby explain infertility under certain conditions, but may also provide a novel approach to male contraception.     

Regulation of TSC2 by 14-3-3 binding

Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a physical and functional complex to regulate cell growth. Recently, it has been demonstrated that TSC1.TSC2 functions to inhibit ribosomal S6 kinase and negatively regulate cell size. TSC2 is negatively regulated by Akt phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3 in vivo. Phosphorylation of Ser(1210) in TSC2 is required for its association with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function of TSC2 and represents a possible mechanism of TSC2 regulation.     

Biochemical characterization of CD39L4

Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.     

Effect of cetrorelix on sperm morphology during migration through the epididymis in the cynomolgus macaque (Macaca fascicularis)

The importance of the cynomolgus monkey as a model for human reproductive medicine prompted this examination of epididymal sperm morphology. Computer-aided sperm morphological analysis was used for the first time to provide morphometric data on sperm heads as they traversed the epididymal duct of Macaca fascicularis. The duct was divided into six regions, starting close to the testis (proximal) and ending close to the vas deferens (distal). To determine the androgen-dependence of the changes, one group of animals received a GnRH-antagonist (Cetrorelix, Asta Medica, Frankfurt, Germany) to induce testicular regression and lower epididymal androgens, while a control group received only vehicle. Epididymides were removed 16 and 25 days after treatment, and sperm heads were analysed by a computer-assisted morphometric analyser. Cluster analysis revealed swollen sperm head cells in proximal regions 1 and 2 of the epididymis, but fewer such forms distally. Normal head shapes became the majority in region 4 and these underwent a gradual but statistically significant decrease in size (area, perimeter, length, width) and shape as they reached the distal regions. In the animals given Cetrorelix, sperm with swollen heads were found more distally than in the controls, although they were also never present in the distal cauda (region 6). Normal heads still became predominant in region 4 after 16 days treatment, and in region 6 after 25 days. The normal forms in the cauda epididymidis of treated animals were significantly larger than cells from control animals. We conclude that epididymal sperm maturation in the monkey is characterised by both a loss of sensitivity to distortion on air-drying, and by a decrease in sperm head size. The former, but not the latter, is attained by sperm in androgen-deficient epididymides from GnRH-antagonist-treated monkeys.