Exogenous sn-1,2-diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets

The ability of exogenous sn-1,2-diacylglycerols and analogs to function as bioregulators of protein kinase C in human platelets was investigated. The activation of protein kinase C in platelets is indicated by specific phosphorylation of a 40,000-dalton protein. Dihexanoylglycerol, dioctanoylglycerol (diC8), didecanoylglycerol, and sn-1-oleoyl-2-acetylglycerol were active in stimulating 40,000-dalton protein phosphorylation. Only a trace of phosphorylation was elicited by dibutyrylglycerol. Phosphorylation was not induced by analogs of diC8 in which an -H, -SH, or -Cl group replaced the free -OH, nor by monoacylglycerols or long chain diacylglycerols. Maximum phosphorylation was induced by dihexanoylglycerol, diC8, and didecanoylglycerol at concentrations from 5 to 20 microM and between 5 and 30 S after exposure of platelets to these diacylglycerols. Under conditions of maximal phosphorylation of the 40,000-dalton protein, these diacylglycerols did not induce phosphatidylinositol turnover, or platelet aggregation, or stimulate release of ATP or serotonin. A small degree of aggregation was evident with platelets isolated in the absence of prostacyclin, and release of serotonin was observed when 1 mM Ca2+ or submaximal concentrations of ionophore A23187 were included. These results are consistent with a model in which platelet activation requires the simultaneous formation of two intracellular signals, diacylglycerols and Ca2+. These diacylglycerols and diacylglycerol analogs provide useful tools to investigate the function of diacylglycerols as bioregulators in intact cells.     

Collagen stimulates [3H]inositol trisphosphate formation in indomethacin-treated human platelets.

The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.     

Estimating body size of fossil sirenians

Estimates of fossil sirenian body size are important for understanding niche partitioning among possibly sympatric species. Because of the paucity of complete fossil skeletons, we explored the utility of three morphometric predictors of body size: (condylobasal skull length [BSL]; occipital condyle width [OCW]; and foramen magnum width [FMW]) in extant sirenians—Florida manatees (Trichechus manatus latirostris) and dugongs (Dugong dugon)—and then applied these to obtain estimates of body size in extinct sirenian taxa. Condylobasal length of the skull is a more accurate predictor of body size in extant Florida manatees and dugongs than are width of the occipital condyles or width of the foramen magnum. Body length (BL) is predicted more accurately than is body weight (BW) for all three morphometric predictors. For our sample of fossil sirenians, BSL, OCW, and FMW were used to generate predicted BLs and BWs. Preliminary assessments of fossil sirenian faunas from Florida and India suggest that body mass could have been one of several possible important morphological parameters accounting for feeding niche separation.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Antisera specific for rap 1 proteins distinguish between processed and nonprocessed rap 1b

Polyclonal antisera were generated against synthetic peptides corresponding to distinct regions of the rap 1 protein sequences. A "rap 1-common" antiserum, prepared against an 18-amino acid segment of the rap 1a protein near the proposed GTP-binding region, reacted with both rap 1a and rap 1b recombinant proteins expressed in Escherichia coli and with two low molecular weight GTP-binding proteins of 22 and 24 kDa in unstimulated human platelets. An antiserum raised against a carboxyl-terminal peptide of rap 1b containing the putative site of post-translational processing reacted strongly with bacterial-expressed recombinant rap 1b and with a 24-kDa GTP-binding protein in platelets, but not with recombinant rap 1a or a 22-kDa GTP-binding protein. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this rap 1b immunoreactive protein coincided with that of bacterial-expressed rap 1b and not with the faster migrating form of rap 1b that incorporates radioactivity from [3H]mevalonic acid in the insect/baculovirus system. This suggests that our rap 1b-specific antiserum recognizes only one form of rap 1b, that which has not undergone carboxyl-terminal post-translational processing.     

THE MUSCULAR HYDROSTAT OF THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS): A FUNCTIONAL MORPHOLOGICAL MODEL OF PERIORAL BRISTLE USE

Facial musculature was examined in the Florida manatee, Tricbecbus manatus latirostris , in order to develop a functional model of perioral bristle use. Muscles identified include the M. levator nasolabialis, M. buccinatorius, M. maxillonasolabialis, M. centralis nasi, M. lateralis nasi, M. spbincter colli profundus pars oris, M. orbicularis oris, M. mandibularis, and M. mentalis. A new muscle, M. centralis nasi , has been named and is an integral part of perioral bristle movement. The snout of the Florida manatee is capable of performing complex movements. The prehensile ability of Florida manatees can be explained in the context of a muscular hydrostat as defined by Kier and Smith (1985). Eversion of certain bristles in the upper lip occurs by shortening longitudinal, transverse, and semicircular muscles in combination with volume displacement due to compensatory changes in the shape of the snout. Midline sweeping of these bristles is accomplished by the contraction of M. centralis nasi. Eversion of bristles on the lower jaw is a result of shortening of M. mentalis. Contraction of M. orbicularis oris pushes vegetation into the oral cavity. All observed movement patterns and uses of perioral bristles can be explained by variation of these sequences within the context of muscular hydrostat function.     

DISTRIBUTION AND INNERVATION OF FACIAL BRISTLES AND HAIRS IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS)

The distribution and anatomy of sirenian perioral bristles (modified vibrissae) and facial hairs are of interest because of their use during feeding and tactile exploration. In the present study we have identified six fields of perioral bristles on the face of the Florida manatee (T. manatus latirostris), four (U1-U4) on each side of the upper lips and oral cavity, and two (L1-L2) on each side of the lower lip pad, inside the oral cavity and rostral to the horny mandibular pad. Each field has a characteristic location, number of bristles, and range of bristle length and diameter. There is a mean of 110 (± 19) bristles per side, with no left-right differences. Branches of the infraorbital nerve innervate the bases of the largest bristles (U2 group) on the upper bristle pad, and the inferior alveolar nerve supplies the bristles of the lower bristle pad. The dorsal and ventral buccal branches of the facial nerve innervate the superficial facial musculature, which is likely to be involved in bristle eversion and other movements which constitute feeding behavior. Hair is denser in the facial region than on the remainder of the body. Within the face, hair is denser on the oral disk than on the supradisk. The oral disk contains bristle-like hair, whereas the supradisk region possesses hair that is similar in length and diameter to that on the postcranial body. The mean total of bristles and hairs per face was 1,942. Means for the subregions were 220 (± 39) bristles on the perioral bristle pads, 601 (± 115) bristlelike hairs in the oral disk region, 710 (± 229) typical hairs in the supradisk region, and 411 (± 108) typical hairs on the chin. There were no significant differences between left and right side counts. Facial hair density was inversely correlated with facial area and body size. These data provide new information on the anatomical basis of the exceptional orofacial activities characteristic of manatees during feeding and tactile exploration.     

PREHENSILE USE OF PERIORAL BRISTLES DURING FEEDING AND ASSOCIATED BEHAVIORS OF THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS)

The use of perioral bristles (modified vibrissae) by 17 captive Florida manatees and approximately 20 wild manatees was analyzed. Captive manatees were fed six species of aquatic vegetation normally eaten in the wild (four freshwater species and two seagrasses). Inanimate objects were placed in the holding tanks with manatees at Lowry Park Zoological Gardens (Tampa, FL) to determine the degree to which perioral bristles were used in exploration and to define the range of manipulative behavior. In addition, behavioral observations were made on the use of perioral bristles during social interactions with conspecifics. Observations were recorded using a Hi8-format video camera. Florida manatees possess an unusually large degree of fine motor control of the snout and perioral bristles. The large and robust perioral bristle fields of the upper lip were used in a prehensile manner during feeding. Bristle use by manatees feeding on submerged vegetation differed from that seen during feeding on floating vegetation. Other behavioral use of the perioral bristles shows variation depending upon the situation encountered. The degree of plasticity of perioral bristle use supports our hypothesis that the vibrissal-muscular complex of the Florida manatee has evolved to increase the efficiency of grazing and browsing on aquatic vegetation and to fully maximize the potential of the manatee as a generalist feeder. The manipulative and sensitive nature of the manatee snout is likely a manifestation of a complex sensory and motor system which has evolved for marine mammal aquatic herbivores living in shallow turbid habitats.