Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands

 Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands.With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay. Accepted: 19 December 1996     

Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats

 The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation. Accepted: 19 February 1997     

Ecological correlates of hind-limb length in the Carnivora

What determines the lengths and proportions of mammalian limbs? While the answer to this question is still largely unknown, a number of workers have recently begun analysing the selection of morphology in a rigorous framework, searching for quantitative links between structure, performance, and their ecological and behavioural context. The present study investigates a variety of ecological and behavioural variables to determine whether or not they are correlated with hind-limb length in the Carnivora. Data were analysed by using phylogenetically independent contrasts and phylogenetic analysis of covariance. We found that traditional perceptions of limb length are often inaccurate; some species widely regarded as relatively long-legged actually have limb lengths near those expected for their body size. Interestingly, relative hind-limb length is not a significant predictor of distance moved daily, home-range area, or prey size. Phylogenetic ANCOVA results, however, indicate a relationship between prey-capture behaviour and relative hind-limb length. These findings suggest that the evolution of carnivoran limb length has been most influenced by selection for prey-capture behaviour. These results, coupled with those of other studies, can be used to suggest which performance variables could most fruitfully be studied in the laboratory to understand the selection of the structure of the mammalian limb. We suggest that relevant performance variables might be: maximum jump height and/or length, the ability to generate outforces, and levels of stress tolerance in limb bones during prey pursuit/capture.     

Almitrine and doxapram decrease fatigue and increase subsequent recovery in isolated rat diaphragm

McGuire, Michelle, Michael F. Carey, and John J. O'Connor.Almitrine and doxapram decrease fatigue and increase subsequent recovery in isolated rat diaphragm. J. Appl.Physiol. 83(1): 52-58, 1997.The effects ofalmitrine bimesylate and doxapram HCl on isometric force produced by invitro rat diaphragm were studied during direct muscle activation at37°C. Doxapram and almitrine ameliorate respiratory failureclinically by indirectly increasing phrenic nerve activity. This studywas carried out to investigate possible direct actions of these agentson the diaphragm before and after fatigue of the fibers. Two age groupsof animals were chosen [6-14 wk (group1) and 50-55 wk (group2)] because it is known that increasing agedecreases a muscle fiber's resistance to fatigue. Muscle strips wereisolated from both group 1 and group 2 and directly stimulated (2-mspulse duration, 5-15 V) to produce twitch tensions of 1.3 and 2.1 N/cm², respectively. At lowconcentrations, doxapram (20 µg/ml) and almitrine (12 µg/ml)had no effect on twitch contraction or 100-Hz tetanic tension. However,40 µg/ml doxapram and 30 µg/ml almitrine increased twitch tensionby 9.0 ± 1.4 and 11.6 ± 1.9%, respectively, in animals ofgroup 2 (n = 5). A fatigue protocol consistingof low-frequency stimulation (30-Hz trains, 250-ms duration every 2 sfor 5 min) caused a reduction of twitch tension in animals ofgroup 1 (48 ± 4% ofcontrol) and group 2 (28 ± 4% ofcontrol). At 90 min postfatigue, the twitch tension recovered to 72 ± 3 and 42 ± 2% of control values ingroup 1 and group2, respectively. In the presence of doxapram (20 µg/ml), there was a significant increase in the recovery of twitchtension at 90 min in group 1 andgroup 2 (84.5 ± 3.2 and 80.1 ± 2.8%, respectively) compared with controls at 90 min postfatigue. Inthe presence of almitrine (12 µg/ml), there was a full recovery fromfatigue in group 1 animals (100% ofcontrol) and a recovery to 95.6 ± 2.1% of control ingroup 2 animals at 90 min. Theseresults demonstrate a significant improvement in the rapidity andmagnitude of recovery from fatigue in the rat diaphragm muscle in thepresence of both doxapram and, especially, almitrine. These effects maybe due to changes in intracellular calcium, ADP/ATP ratios, or oxygenfree radical scavenging.

     

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

The role of the basement membrane in differential expression of keratin proteins in epithelial cells

Extracellular matrix is considered to play an important role in determining the phenotype of cells with which it interacts. Here we have investigated the possibility that extracellular matrix is involved in specifying the pattern of keratin expression in epithelial cells. For these studies, we have developed an explant system in which epithelial cells from one type of stratified epithelial tissue, namely conjunctiva, are maintained on an extracellular matrix substrate derived from a different tissue, namely cornea. These ocular tissues are ideal for such analyses since they express distinct sets of keratins. For example, bovine conjunctival epithelium processed for immunofluorescence is not recognized by antibody preparations against keratin K3 or K12. In contrast, K3 and K12 antibodies generate intense staining in bovine corneal epithelium. At the immunochemical level, conjunctival cells in situ appear to possess no K12 and only trace amounts of K3, whereas corneal epithelial cells in situ possess both K3 and K12. When conjunctival cells are maintained on a corneal substrate with an intact basement membrane for 10 days in vitro they begin to express keratin K12 as determined by immunofluorescence. On the other hand, conjunctival cells that are maintained on a corneal substrate lacking a basement membrane fail to show staining with K12 antibodies. Conjunctival cells begin to show intense staining using K3 antibodies within about 10 days of being placed in culture regardless of their substrate. These results indicate that basement membrane can play a positive role in determining cell-specific expression of certain keratins such as K12. However, other keratins such as K3 may be "unmasked" and/or their expression may be upregulated simply by placing conjunctival epithelial cells in culture. We speculate that in conjunctiva K3 expression is influenced by certain negative exogenous factors. We discuss the possible means of regulation of keratin expression in our model system.     

Urinary zinc and its relationships with microalbuminuria in type I diabetics

We investigated whether zincuria is associated with microalbuminuria in type I (insulin-dependent) diabetics (IDDM). In 169 IDDM, 215 overnight urine samples were collected for simultaneous assay of zinc and albumin. In 76 samples with excessive microalbuminuria (>15 mg/L), zincuria was higher than in the 139 other samples (0.83±0.06 vs 0.58±0.03 mg/Lp<0.001), though zincuria and microalbuminuria were not significantly correlated. An exercise provocation test was performed in 78 IDDM. Although microalbuminuria increased, zincuria did not change during the test. Another group of 83 IDDM underwent urinary zinc determination over a period of 1 h of recumbency. The 48 patients who had a zincuria higher than the mean+2 SD of control values had higher microalbuminuria at rest (48±16 μg/min vs 12±2p<0.01) and after exercise (111±33 vs 42±14p<0.02) than the remaining 35 subjects. Both subgroups did not differ for zinc intake and zincemia. Thus, incipient nephropathy as detected by the measurement of microalbuminuria is associated with a highly significant increase in zinc excretion, which is not proportional to albumin leakage, nor is it amplified during exercise. Hyperzincuria is not explained by an increase in zinc intake and does not result in hypozincemia.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Zooplankton impacts on chlorophyll and transparency in Onondaga Lake,New York,USA

The transparency of polluted, hypereutrophic Onondaga Lake, New York, USA has improved substantially in the late 1980's as a result of reductions in phytoplankton biomass, in the absence of significant reductions in external phosphorus loading. Much of this improvement has been due to the occurrence of clearing events, e.g. sudden and dramatic increases in transparency. Field measurements, laboratory experiments, and modelling analyses were utilized to identify processes regulating phytoplankton standing crop during the spring to fall interval of 1987. Changes in the zooplankton community documented over the past decade support the conclusion that increased zooplankton grazing has contributed to improvements in transparency. Herbivores now represent a greater fraction of the zooplankton population and more efficient cladocerans are present in greater numbers. Biomanipulation practices, e.g. reestablishment of piscivorous species, designed to reduce the abundance of planktivorous fish species in Onondaga Lake, may serve to reduce pressure on the grazing community and thus result in further improvements in transparency.