Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells.

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.

Activation of exocytosis by GTP analogues in adrenal chromaffin cells revealed by patch-clamp capacitance measurement.

The role of GTP-binding proteins in exocytosis in bovine adrenal chromaffin cells was examined using patch-clamp capacitance measurement. Internal dialysis with the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate and xanthosine triphosphate (XTP) activated a capacitance increase. Exocytosis triggered by XTP was blocked by guanosine 5'-[beta-thio]diphosphate (GDP beta S) but Ca(2+)-induced exocytosis was unaffected. The capacitance increase due to XTP could not be explained by Ca2+ mobilisation since Ins(1,4,5)P3 and caffeine did not mimic the response. Chromaffin cells appear to possess a Ca(2+)-independent pathway for exocytosis that involves GTP-binding proteins. The magnitude of the response to XTP suggested that GTP analogues stimulate both exocytosis and recruitment of secretory granules.

"Giant" muscle fibres in skeletal muscle of normal pigs.

Observations made during a growth and development study of the semitendinosus and trapezius muscles of 49 purebred Large White pigs between birth and 128 days of age revealed the presence of giant fibres. The occurrence, histochemical and ultrastructural properties of these giant fibres were investigated. A high proportion of the pigs (85 per cent) contained giant fibres in their muscles but these giant fibres usually represented less than 1 per cent of the total myofibre population. Giant fibres possessed enhanced adenosine triphosphatase activity and a high capacity for oxidative metabolism (indicated by succinate dehydrogenase activity) which was reflected ultrastructurally by the greatly heightened electron density of myofibrils and by an abnormally high percentage of mitochondria and lipid droplets. These deviations from normal muscle fibre composition, together with the reduced percentage volume of sarcoplasmic reticulum, were consistent with changes seen in functionally over-loaded muscle. It appears that giant fibre anomalies occur through increased activity stimulated in occasional muscle fibres, perhaps by a structural defect, such as an inadequate amount of sarcoplasmic reticulum, which causes hyper-contractile activity within the fibres and associated compensatory adaptations. Giant fibres did not appear to represent fibres undergoing degenerative changes.

A comparative evaluation of the in vitro penetration performance of the improved Crest complete toothbrush versus the Current Crest complete toothbrush, the Colgate Precision toothbrush and the Oral-B P40 toothbrush.

Removal of plaque and debris from interproximal surfaces during toothbrushing has generally been difficult to achieve, in large part because traditional flat-bristled toothbrushes do not offer good interproximal penetration. As a result, a number of varying bristle designs have been developed, with the rippled-design brush shown to be particularly effective at removing interproximal plaque. Recently, an existing brush, the original Crest Complete, was modified to offer a more deeply rippled version. This study evaluated the interproximal penetration of four bristle designs: rippled pattern (original Crest Complete), deeper rippled pattern (improved Crest Complete), multi-level (Colgate Precision), and flat-tufted (Oral-B P40). The study used a previously reported in vitro model for determining interproximal penetration of manual toothbrushes (J Clin Dent 5:27-33, 1994). In order to effectively mimic the in-use characteristics of toothbrushing, this model is based on analysis of videotaped consumer brushing habits, tooth morphology, and in vivo plaque tenacity characteristics and uses the three most predominantly used brushing techniques (circular, up-and-down, and back-and-forth, with the brush held at both 45 and 90 degrees to the tooth surface). In addition, the model's brush stroke length, brush force, and brush speed are likewise based on analysis of consumer brushing patterns. The results of the study indicate that the new Crest Complete with deeper rippled bristles provided significantly superior (p < or = 0.05) interproximal penetration than the Colgate Precision and Oral-B brushes overall and for three of the four brush strokes tested. In addition, the new Crest Complete was found to provide significantly superior interproximal penetration to the original Crest Complete overall and in circular and up-and-down strokes, and the original Crest Complete provided superior overall interproximal penetration to the Colgate and Oral-B brushes.

The numbers and types of muscle fibres in large and small breeds of pigs.

M. semitendinosus and m. trapezius (portion) were removed from eight miniature pigs ranging from 21 to 160 days of age and eight age-control as well as eight weight-control commercial Large White pigs. Complete transverse frozen sections were obtained for each muscle sample and stained for various enzyme activities including myosin adenosine triphosphatase activity which enabled the identification of 'metabolic bundles'. This in turn enabled conclusions to be made about the prenatal development of the muscle in terms of primary and secondary myofibres. The Large White pigs contained 173% more muscle fibres in m. semitendinosus than did the miniature pigs. Primary myofibre number was found to be about four times more important than secondary to primary myofibre ratios in determining myofibre number in the two breeds of pigs. Both primary myofibre number and secondary to primary myofibre ratios were, however, significantly greater in Large White than in miniature pigs. When the age- and weight-control Large Whites were compared with the miniature pigs it was found that at any given live weight the miniature pigs had thicker myofibres whereas at the same age there was no significant difference. The total area of m. semitendinosus occupied by slow myofibres was about three times greater in the Large White pigs; the functional aspects of this are discussed. It was concluded that genetically smaller animals develop fewer muscle fibres in their muscles by a different mechanism to that exhibited by animals which are smaller due to nutritional deprivation in utero.

The effects of low birthweight on the ultrastructural development of two myofibre types in the pig.

An ultrastructural investigation of the postnatal development of oxidative and non-oxidative fibres from the deep and superficial portions of the semitendinosus muscle, respectively, was undertaken on 32 pure bred Large White pigs from a total of 11 litters. This study quantifies the changes in mitochondrial, lipid droplet and myofibrillar content of these two myofibre types between birth and 84 days of age, and evaluates differences between the largest male (mean birthweight of 1559 g), smallest normal male (1147 g), and runt (758 g) littermates. The oxidative and non-oxidative fibres, as well as possessing different complements of mitochondria, lipid droplets and myofibrils, showed different rates of myofibrillar accumulation. The relatively small postnatal change in the percentage volume of myofibrils of oxidative fibres, as opposed to the high change within the non-oxidative fibres, presented a cytological basis by which to explain the differential effects of growth retardation on these fibre types. The ultrastructural composition of myofibres was not impaired by reduced birthweight except when, as in two extreme cases, birthweight was severely reduced. In these instances the myofibrillar percentage volume of the non-oxidative fibres was greatly affected.

The growth and differentiation of porcine skeletal muscle fibre types and the influence of birthweight.

Muscle growth and development was studied in 49 Large White pigs from a total of 17 litters. Representative large (mean birthweight of 1544 g), small (1144 g) and runt (776 g) littermates were selected and slaughtered at the same age, ages ranging from birth to 128 days. Fresh frozen, serial transverse sections taken from the semi-tendinosus and trapezius muscles of these animals were stained for the histochemical demonstration of acid and alkaline pre-incubated adenosine triphosphatase, succinate dehydrogenase and glycogen phosphorylase. Profiles of the muscle fibre types were compiled for each animal. In both muscles the number of slow oxidative (SO) fibres, that were arranged together in groups within 'metabolic bundles', increased with growth. The transverse sectional area (TSA) of the semitendinosus muscle increased with the 2/3 power of liveweight whereas the area occupied by SO fibres increased at a rate significantly greater than 1.0 (P less than 0.01). Regression analysis revealed that the area of this muscle occupied by SO fibres was greater (P less than 0.001) in runt and small littermates relative to their large littermates when they were compared at an equal liveweight. This greater TSA of the semitendinosus classified as 'SO' in lower birthweight pigs was the result of a combination of higher percentages (P less than 0.05) of SO fibres and significantly greater (P less than 0.001) SO fibre mean TSAs. The mean TSAs of all myofibre types were similar between littermates of the same age but most types were of greater TSA in the lower birthweight littermates when compared (by regression analysis) at the same liveweight suggesting that fibre TSA was age- rather than weight-related. The higher percentage of SO fibres in the low birthweight pigs, when compared at an equivalent liveweight to their large littermates, appeared to be related to their affected secondary/primary fibre number ratio. This phenomenon, plus the data on the number of slow fibres per metabolic bundle, indicated that it was apparently the number of slow fibres per metabolic bundle which was regulated with liveweight gain rather than the resultant percentage of slow fibres within the muscle.

Microinjection of covalently cross-linked actin oligomers causes disruption of existing actin filament architecture in PtK2 cells.

Experiments were performed to determine the effects of interrupting the flux of actin monomers between unpolymerised and polymerised pools in PtK2 cells by (1) microinjecting exogenous polymerisation nuclei and (2) blocking endogenous assembly sites with low concentrations of cytochalasin D. Fluorescent actin oligomers were prepared by glutaraldehyde cross-linking F-actin derivatised at cysteine-374 with 5-iodoacetamido-fluorescein. These oligomers caused rapid nucleation of polymerisation of pyrene-labelled actin in vitro. Different numbers of polymerisation nuclei were injected into PtK2 cells and the cells were incubated for various times. Microinjection of between 1.2 X 10(4) and 1.8 X 10(4) nuclei per cell resulted in the complete disassembly of existing actin filament structures in nearly half of the cells within 15 min. Existing structures in such cells were replaced by foci of polymerised actin, which co-localised with concentrations of nuclei. Injection of increasing numbers of nuclei between 3 X 10(3) and 1.2 X 10(4) caused fragmentation of stress fibres in an increasing proportion of cells, whereas injection of less than 3 X 10(3) caused no obvious effects even over a 90 min incubation period. These data indicate that the degree of disruption of stress fibres was a function of the number of nuclei injected, but that it was less dependent on the incubation time. The minimum number of injected nuclei causing complete disruption of actin filament structures provides an estimate for the number of endogenous nuclei involved in filament turnover, whereas the minimum period for reorganisation (about 15min) implies a maximum time for the complete turnover of actin in the system.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells.

The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of alpha-, beta- and gamma-caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of alpha- and gamma-caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mammary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of beta- and gamma- but not alpha-caseins. These results suggest that phosphorylation of alpha-casein normally occurs in Golgi cisternae whereas that of beta- and gamma-caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.

The nature and regulation of actin filament turnover in cells.

Actin filaments in mammalian cells form a number of different architectures in conjunction with a number of different actin-binding proteins. In motile cells these complex architectural arrangements of actin filaments and associated proteins continuously adjust their 3-dimensional organisation to modify the shape and behaviour of cells in response to external information. Microinjection experiments with fluorescently-labelled actin monomers suggest that there is a continual exchange of monomers between the actin filaments and a soluble pool such that individual monomers exist for only a few minutes within polymers. These data suggest that remodelling of the actin filament architectures occurs by the continuous assembly of new filaments which is balanced by the disassembly of obsolete structures. The mechanisms driving and regulating the assembly and disassembly cycle are not yet clearly understood. The properties of the actin assembly ATPase in vitro suggest that the intrinsic exchange of monomers between polymers and the monomer pool is driven by the stoichiometric ATP hydrolysis which is uncoupled from monomer addition and leads to both treadmilling and to the potential for mechanisms analogous to the dynamic instability models proposed for microtubules. Because of the relatively rapid rate of ATP hydrolysis by monomers in the filament (k = 0.05-0.02/s), it is assumed that most of the F-actin in cells is in its ADP form. ADP-F-actin binds inorganic phosphate with a Kd close to that of cytoplasmic concentrations to form an ADP.Pi-F-actin form which has different kinetic, structural and behavioural properties to ADP-F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells.

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

Annexin II (calpactin I) in the mouse mammary gland: immunolocalization by light- and electron microscopy.

To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalization in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extensions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vesicles could clearly be identified as casein-containing vesicles while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma-isoforms of actin.

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

Inhibition of constitutive protein secretion from lactating mouse mammary epithelial cells by FIL (feedback inhibitor of lactation), a secreted milk protein.

The effect of a protein feedback inhibitor of lactation (FIL) on casein synthesis and secretion was examined using isolated acini from lactating mouse mammary gland. As previously found, FIL partially inhibited protein synthesis but produced an additional inhibition of constitutive casein secretion. The inhibition of synthesis and secretion showed similar dose-dependency and the inhibition was fully reversible. Constitutive secretion of pre-formed protein was inhibited by FIL in a pulse-chase protocol, indicating that the inhibitor regulated protein secretion by reducing protein movement through the secretory pathway independently of any initial inhibition of synthesis. Regulated exocytosis was not inhibited since casein release due to elevation of cytosolic Ca2+ concentration by the ionophore ionomycin was unaffected. Brefeldin A, which is known to block ER-to-Golgi transport, also inhibited both protein synthesis and secretion in mammary cells. The action of FIL on synthesis and secretion and previously described actions on casein degradation would be consistent with a block at an early stage in the secretory pathway. In support of this idea FIL treatment was found to result in vesiculation and swelling of the endoplasmic reticulum. These data provide evidence for a novel control of a constitutive secretory pathway by a physiological extracellular regulatory protein.

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin.

Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against alpha- and gamma-actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the "sarcomeric" proteins alpha-actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of "sarcomeric" proteins in regard to myofibrillogenesis.