The distribution of intracellular ions in the avian salt gland.

To investigate the mechanism of salt secretion in the avian salt gland, we used quantitative electron probe microanalysis to measure the intracellular elemental concentrations in dry cryosections of unspecialized and partially specialized secretory epithelial cells from fresh water- and salt water-adapted ducklings, respectively. In conjunction with this, human and duckling erythrocytes were also analyzed, since these provided the experimental basis for using in situ erythrocytes as standards for determining the local water content of epithelia from the analysis of dried cryosections. The microprobe results from both types of erythrocytes compared favorably with chemical determinations of elemental concentrations. The nucleated avian erythrocytes, whose wet-weight elemental concentrations were determined by a compartmental analysis that required neither a peripheral standard nor a measure of the local mass, revealed a marked accumulation of P and K in the nucleus (388 and 190 mmol/kg wet wt, respectively) relative to the cytoplasm (67 and 85 mmol/kg wet wt). In both developmental states of the epithelial cells, the nucleus and apical cytoplasm had essentially similar and unremarkable concentrations of Na (76 and 83 mmol/kg dry wt, respectively, in the adapted cells vs. 72 and 81 mmol/kg dry wt in the control cells) and K (602 and 423 mmol/kg dry wt vs. 451 and 442 mmol/kg dry wt). Chloride, however, which was in general rather high, was significantly depressed in the apical cytoplasm of adapted cells only (164 and 124 mmol/kg dry wt in the nucleus and cytoplasm, respectively, of adapted cells (P less than 0.05) vs. 138 and 157 mmol/kg dry wt for control cells (P less than 0.05). Cation concentrations (Na + K) were elevated approximately 15% in the basal regions of adapted cells as compared with apical cytoplasm. When tissue water variations are accounted for, the results suggest that: (a) an active, energy-requiring process is responsible for chloride accumulation in this cell; (b) the apical membrane is a regulatory site for secretion; and (c) there are regional distinctions in the distribution of ions and water, particularly in the salt water-adapted cell. These conclusions are consistent with active chloride transport as the basis for salt secretion in this tissue.

Effects of manganese on Streptococcus mutans planktonic and biofilm growth.

Streptococcus mutans, an agent of dental caries, was tested for growth in the presence or absence of manganese (Mn), since studies have linked Mn levels with cariogenic potential. Seven S. mutans serotype c strains were grown in chemically defined medium under different atmospheric conditions: 5% CO2, O2-enriched 5% CO2 (shaking) and anaerobic. There was significant strain variability with respect to Mn requirements under the various conditions tested. Both sucrose-dependent and sucrose-independent biofilm growth by strain UA159 were affected by the absence of Mn. S. mutans strains show highly variable responses to both high and low Mn concentrations.

A morphological and phenotypic analysis of Walker 256 cells.

A detailed morphological analysis of Walker 256 cells sensitive and resistant to cis-diamminedichloroplatinum(II) has been performed. Two cell populations are identified by electron microscopy of differing differentiation corresponding structurally to cells reported in experimentally induced metastases. Phenotyping of the cells using a number of monoclonal antibodies by immunocytochemistry and flow cytometry showed the absence of epithelial cell markers: however, the cells stained intensely for markers for germ and/or hematopoietic cells. Further studies utilizing monoclonal antibodies to lymphoid, myeloid, and monocytoid cells showed the cells to be monocytoid in origin. No evidence of cell heterogeneity was evident from the phenotypic experiments (a biphasic pattern was not observed). Enzyme histochemistry showed strong focal acid phosphatase activity suggestive of cells of hematopoietic origin. Thus the concept that these cells reflect an epithelial cell of origin is not substantiated by phenotyping with two methodologies.

The Meissner corpuscle revised: a multiafferented mechanoreceptor with nociceptor immunochemical properties.

Meissner corpuscles (MCs) in the glabrous skin of monkey digits have at least three types of innervation as revealed by immunofluorescence. The previously well known Aalphabeta-fiber terminals are closely intertwined with endings from peptidergic C-fibers. These intertwined endings are segregated into zones that alternate with zones containing a third type of ending supplied by nonpeptidergic C-fibers. Although MCs are widely regarded as low-threshold mechanoreceptors, all three types of innervation express immunochemical properties associated with nociception. The peptidergic C-fiber endings have readily detectable levels of immunoreactivity (IR) for calcitonin gene-related peptide (CGRP) and substance P (SP). The Aalphabeta endings have relatively lower levels of IR for CGRP and SP as well as the SP neurokinin 1 receptor and vanilloid-like receptor 1. Both the Aalphabeta and peptidergic C-fiber endings were also labeled with antibodies for different combinations of adrenergic, opioid, and purinergic receptors. The nonpeptidergic C-fiber endings express IR for vanilloid receptor 1, which has also been implicated in nociception. Thus, MCs are multiafferented receptor organs that may have nociceptive capabilities in addition to being low-threshold mechanoreceptors.

High-affinity binding of fibronectin to cultured Kupffer cells.

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative "fibronectin receptors" per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

Neurofilament distribution is altered in the Mnd (motor neuron degeneration) mouse.

Motor neuron degeneration (Mnd) is a genetic neurodegenerative disease of the mouse that is characterized by a progressive increase in motor dysfunction, moving from hind to fore limbs, leading to paralysis. An immunocytochemical analysis of the neurofilament distribution in spinal motor neurons in Mnd mice from all stages of the disease, including the presymptomatic, was performed using antibodies to different neurofilament subunits with different degrees of phosphorylation. Perikarya that stained with antibodies to phosphorylated neurofilaments were present in Mnd and control spinal cords, but the number of stained perikarya in Mnd was not significantly different from controls. There was a marked redistribution of neurofilaments within the cytoplasm of some motor neurons in Mnd cords. In Mnd but not controls, the immunoreaction product appeared marginated, leaving areas in the cytoplasm absent of immunostaining. These areas were observed in all stages of the disease, but less predictably in presymptomatics. Both the size of the areas and the number of motoneurons containing these areas appeared to increase with the severity of the disease. The number of anterior horn neurons in the hind limb region of lamina IX in spinal segment L4 of Mnd was lower than in controls, suggesting there is a loss of neurons in Mnd.

Characterization of corticotropin-releasing hormone (CRH) in human skin.

We have confirmed the expression of CRH and CRH receptor type 1 genes in human skin, cultured HaCaT keratinocytes, squamous cell carcinoma, and melanoma cells. The size of CRH messenger ribonucleic acid (mRNA), estimated by Northern blot hybridization, was 1.5 kilobases. CRH peptide was identified by reverse phase high pressure liquid chromatography separation in both whole skin and cultured cells. Forskolin and dexamethasone at concentrations of 10 micromol/L stimulated and inhibited, respectively, CRH peptide production in squamous cell carcinoma and melanoma cells, but had no significant effect on the CRH mRNA level. In melanoma cells, stimulation of melanogenesis down-regulated CRH receptor type 1 mRNA expression, but was without effect on CRH mRNA production. We suggest that in human skin the CRH signaling system is both operative and under regulatory control.

Chimerasome-mediated gene transfer in vitro and in vivo.

Proteoliposome delivery vesicles can be prepared by the protein-cochleate method [Gould-Fogerite and Mannino, Anal. Biochem. 148 (1985) 15-25; Mannino and Gould-Fogerite, Biotechniques 6 (1988) 682-690]. Proteins which mediate the entry of enveloped viruses into cells are integrated in the lipid bilayer, and materials are encapsulated at high efficiency within the aqueous interior of these vesicles. We describe proteoliposome-mediated delivery of proteins and drugs into entire populations of cells in culture. Material can be delivered gradually by Sendai-virus-glycoprotein-containing proteoliposomes. Alternatively, synchronous delivery to a population can be achieved by exposing cell-bound influenza glycoprotein vesicles briefly to low pH buffer. When DNA is encapsulated, chimeric proteoliposome gene-transfer vesicles (chimerasomes), which mediate high-efficiency gene transfer in vitro and in vivo, are produced. Stable expression of a bovine papilloma virus-based plasmid in tissue-cultured cells, at 100,000 times greater efficiency than Ca.phosphate precipitation of DNA, with respect to the quantity of DNA used, has been achieved. Stable gene transfer and expression in mice has been obtained by subcutaneous injection of chimerasomes containing a plasmid expressing the early region of polyoma virus. In one experimental group, 50% of the mice developed tumors which were shown to express polyoma virus early proteins and contain the transferred DNA. This is the first report of stable gene transfer in animals mediated by a liposome- or proteoliposome-based system.

Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H(2)O(2).

Manganese superoxide dismutase (MnSOD) overexpression has been shown to reverse the malignant phenotype in a variety of tumor cell lines. The inhibition of proliferation and reversal of the malignant phenotype has been attributed to an increase in H(2)O(2) production as a result of the dismutation reaction. However, direct evidence in support of this hypothesis has not been forthcoming. To evaluate the contribution of H(2)O(2) in the regulation of cell growth in response to MnSOD overexpression, control and MnSOD-overexpressing HT-1080 fibrosarcoma cells were transfected with constructs that direct catalase to either the mitochondrial or cytosolic compartments. Overexpression of catalase in either compartment reversed the proliferative and clonogenic inhibition associated with MnSOD overexpression, blocked the increase in the steady state levels of H(2)O(2) as measured by flow cytometric analysis of 2', 7'-dichlorofluorescein diacetate, and increased protection from the cytotoxicity of H(2)O(2). In addition, mitochondrial or cytosolic catalase enhances respiration through complex I and II in both control and MnSOD overexpressing cell lines and reverses a MnSOD-dependent decrease in net ATP production. Thus, catalase reverses the proliferative inhibition associated with MnSOD overexpression and may also play an important role in metabolic regulation.

Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin.

Evidence is provided that human skin, the largest body organ exposed to multiple stressors, expresses proopiomelanocortin (POMC), corticotropin releasing hormone (CRH) and CRH-receptor (CRHR) genes in vivo. In vitro studies show that POMC and CRHR mRNAs are transcribed in melanocytes, cells derived from the neural crest, and in keratinocytes, cells derived from the ectoderm. CRH mRNA is transcribed in cultured melanocytes but not in keratinocytes. It is proposed that an equivalent of the 'hypothalamus-pituitary axis' composed of the CRH-CRHR-POMC loop is conserved in mammalian skin.

16S mitochondrial ribosomal RNA degradation is associated with apoptosis.

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.

An in vitro model for studying the contributions of the Streptococcus mutans glucan-binding protein A to biofilm structure.

The method described here for analyzing biofilms was sensitive enough to allow the detection of differences formed by pure cultures of S. mutans or a GbpA knockout strain. Other strains have also been tested, and the differences in biofilm structure were sometimes even more extensive (data not shown). The advantages of this method are that it is quick, inexpensive, and adaptable to almost any laboratory setting. The constant rotation of the cultures, which was employed to simulate salivary flow, appears to be a critical element for establishing biofilm differences. An analysis of protein profiles confirmed that the biofilm bacteria were metabolically distinct from the planktonic phase bacteria. For the strains tested, the variations in biofilm architecture could be visualized with or without magnification. Staining of the bacteria was not required, though we typically stained the biofilms with either crystal violet or Schiff's reagent. Altogether, this in vitro method for generating biofilms allowed the evaluation of visual, quantitative (confocal microscopy), and functional (antimicrobial susceptibility) differences. We have employed these methods in a reductionist approach to understanding the contribution of individual proteins to dental plaque development. These methods may also be useful in the screening of mutants that would be of greatest for testing in multispecies biofilms, animal models, or more complex biofilm models.

Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells.

Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+ in the presence of a monospecific rabbit IgG fraction directed against bovine dopamine beta-hydroxylase. The anti-dopamine beta-hydroxylase was labeled either with fluorescent protein A or with a fluorescent second antibody to rabbit IgG. Stimulation produced a patchy cell surface distribution of fluorescence. There was no noticeable internalization of the fluorescence for up to 2 h. In similar experiments using fluorescent monovalent fragments (Fab) of the same monospecificidopamine-beta-hydroxylase IgG, a more uniform distribution of the fluorescence was observed. A few min after a 5 min period of stimulation with Ba2+, the fluorescence appeared to be on or near the cell surface; however, after 20 min or more it was distributed throughout the cytoplasm except that the cell nuclei were not labeled. Thus, dopamine beta-hydroxylase which appeared on the cell surface as a consequence of exocytosis was internalized in the presence of monovalent antibody fragments, but not in the presence of the divalent (polyclonal) antibody, presumably because endocytosis of dopamine beta-hydroxylase was inhibited by crosslinking of the dopamine beta-hydroxylase molecules. The internalized anti-dopamine beta-hydroxylase Fab fragments were found to reappear on the cell surface during a second secretory response. It is concluded that the interior of the chromaffin granule membrane, for which dopamine beta-hydroxylase is a marker, becomes exposed on the surface of the cell during secretion and that the membrane is then retrieved back into the cell where it can be re-used in a further secretory cycle.

Spatiotemporal expression, distribution, and processing of POMC and POMC-derived peptides in murine skin.

In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides beta-endorphin, ACTH, beta-MSH, and alpha-MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, beta-endorphin, beta-MSH, and alpha-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin.

A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.

The presence of Fe(CN)6(-4) provides sequential, one-electron reduction pathways for OSO4. An equilibrium is established containing OSO4, Fe(CN)6(-4), Fe(CN)6(-3), OSO2(OH)4(-4), and labile cyano-bridged OS-Fe species containing Os in nominal oxidation states of VIII, VII, and VI. These osmium complexes are chelated by appropriately placed donor atoms in the macromolecular tissue matrix, and chelation facilitates the reduction of osmium in situ to lower oxidation states (predominantly IV) that are relatively nonlabile. The greater reactivity and concentration of the Os(VII and VI) intermediates in this system leads to more Os deposition than OsO4 alone; the chelation is responsible for the immobilization of Os and the observed staining pattern in electron micrographs. Chemical data from model systems and electron micrographs of tissue are presented in support of this mechanism.

Cytochemical demonstration of sodium, potassium-adenosine triphosphatase by a hemepeptide derivative of ouabain.

A cytochemical probe for the ultrastructural localization of the NaKATPase was devised, which utilizes the biological affinity of the noncompetitive inhibitor, ouabain (ouab) to which was coupled a hemepeptide (H11P) which possesses peroxidatic activity. The conjugate, ouab-H11P, had an apparent Ki of approximately 8 x 10(-7) M. When reacted with fixed tissue from the salt gland of osmotically stressed ducklings, the NaKATPase was localized to the basal and lateral infoldings of the plasma membranes of secretory epithelial cells. Reaction product consisted of fine textured deposits distributed in focal patches on the outer aspects of the membrane. Apical membranes were negative, as were intracellular membrane components. Preincubation of tissue with unlabeled ouabain or binding of ouab-H11P in the presence of 10 mM K+, no ATP and no Mg++, resulted in the absence or diminution of reaction product.

Expression of proopiomelanocortin (POMC)-derived melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) peptides in skin of basal cell carcinoma patients.

We proposed that local expression and production of proopiomelanocortin (POMC) peptides may play a role in human skin physiology and pathology, including the development and progression of skin cancers. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting hybridization techniques were used to study gene expression. Reversed-phase (RP) high-pressure liquid chromatography (HPLC) separation with subsequent radioimmunoassays were used to identify alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) peptides. Immunocytochemistry (IHC) was used to localize ACTH, alpha-MSH, and beta-MSH antigens in skin. RT-PCR, RP-HPLC, and IHC analyses documented the expression of POMC mRNA and production of ACTH and alpha-MSH peptides in lesional and perilesional skin of basal cell carcinoma (BCC) patients and in cultured keratinocytes, which was accompanied by the expression of the MC1-R gene encoding the receptor activated by MSH and ACTH. Thirty specimens were analyzed by IHC. Immunoreactive alpha-MSH, beta-MSH, and ACTH were detected, in 21 of 21, in 11 of 20, and in 6 of 8 of lesional skin, and in 6 of 6, in 5 of 7, and in 6 of 8 perilesional skin specimens analyzed, respectively. Antigen distribution was heterogenous and present in BCC, epidermis, hair follicles, dermal mononuclear cells, and extracellular matrix. We conclude that messenger RNA for POMC, MC1-R, and the peptides MSH and ACTH are produced in skin of BCC patients. Because keratinocytes are a target for MSH and ACTH bioregulation, the production of these peptides is stimulated by UVB, and the peptides can act as immunosupressors, we suggest that MSH and ACTH may facilitate development of BCC.

Cytochemical localization of acetylcholine receptors at the neuromuscular junction by means of horseradish peroxidase-labeled alpha-bungarotoxin.

The cytochemical localization of acetylcholine (ACh) receptors at the neuromuscular junction was investigated with a procedure utilizing alpha-bungarotoxin (alpha-BtX) labeled directly with horseradish peroxidase (HRP). Following incubation of tissues in the conjugate and reaction for peroxidase, activity was observed on the junctional folds of the motor endplate. A uniform layer of reaction product approximately 15 nm thick occurred over the apical portions of the junctional folds. Membranes at the bases of the synaptic cleft showed only small amounts of reaction product. Non-junctional regions of the muscle fiber were unreactive. Activity was also observed in the membrane of the axon facing the muscle surface, often including the axolemma overlying the "active zones" of the nerve terminal. Such presynaptic activity was still evident on nerve terminals disjuncted from the synapse by enzymatic treatment prior to incubation in the conjugate. This localization indicates the possible presence of presynaptic ACh receptors within the axolemma. In muscle denervated for 7-12 days, motor endplates were reactive and parajunctional sarcolemma showed slight activity, but most extrajunctional regions contained no obvious accumulations of reaction product. Activity at all sites was prevented by preincubation of tissues in native alpha-BTX prior to incubation in the conjugate and reaction for HRP. This procedure represents a simple and convenient method for the high resolution localization of ACh receptors.

Ubiquitin deposits are present in spinal motor neurons in all stages of the disease in the motor neuron degeneration (Mnd) mutant of the mouse.

Monoclonal antibodies to ubiquitin were used in an immunocytochemical analysis of spinal cord from the Mnd (motor neuron degeneration) mouse, an animal model for motor neuron disease. Tissue from mice with mild, moderate and severe disease, from presymptomatic mice, and age-matched controls were analyzed. Ubiquitin deposits were observed in spinal neurons from presymptomatic animals and all stages of the disease. No immunoreactive deposits were observed in control mice at the concentration of the antibodies used. The presence of ubiquitin immunopositivity in presymptomatic spinal motor neurons suggests that ubiquitination might play a primary role in the pathogenesis of motor neuron disease in the Mnd mouse, and perhaps of motor neuron disease in general.

Cytoplasmic inclusions in spinal neurons of the motor neuron degeneration (Mnd) mouse. I. Light microscopic analysis.

The motor neuron degeneration (Mnd) mutation in the mouse is a late onset, autosomal dominant, neurodegenerative disease in which ventral horn neurons have been shown to contain numerous, large cytoplasmic inclusions. Histochemical and immunocytochemical studies performed on spinal cord from Mnd/Mnd mice in late stages of the disease showed the inclusions to contain protein, lipid and carbohydrate moieties. Spinal neurons, especially those in spinal lamina IX, contained increased beta-glucuronidase activity in the form of large cytoplasmic inclusions. Such inclusions also contained increased acid phosphatase and trimetaphosphatase activity. When immunostained with antiubiquitin antibodies, intracellular ubiquitin deposits were present as accumulations of varying size; some were amorphous while others contained small granules. Extraneuronal ubiquitin deposits were detected in the neuropil. Immunostaining with monoclonal antibody ML30, used here to assay for the presence of a mitochondrial epitope in the inclusions, was widespread and punctate in white and grey matter from Mnd/Mnd and age-matched control spinal cords. The overall pattern of staining was similar for both tissue sources and did not correspond to any of the other probes which reacted with the inclusions in Mnd neurons. The presence of increased levels of lysosomal hydrolases and ubiquitinated molecules suggests that the two general systems for intracellular digestion are activated in Mnd/Mnd spinal neurons.