Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind ¹²⁵I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of ¹²⁵I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When ¹²⁵I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000–160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells. © 1994 Wiley-Liss, Inc.     

Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus

Background: The evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology. The current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved from an archaebacterial ancestor. Recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, particularly regarding the origin of eukaryotic cells.Results We have used sequences of 70 kD heat shock protein (hsp70) — the most conserved protein found to date in all species — to examine the evolutionary relationship between various species. We have obtained two new archaebacterial hsp70 sequences from the species, Thermoplasma acidophilum and Halobacterium cutirubrum. A global comparison of hsp70 sequences, including our two new sequences, shows that all known archaebacterial homologs share a number of sequence signatures with the Gram-positive group of bacteria that are not found in any other prokaryotic or eukaryotic species. In contrast, the eukaryotic homologs are shown to share a number of unique sequence features with the Gram-negative bacteria that are not present in any archaebacteria. Detailed phylogenetic analyses of hsp70 sequences strongly support a specific evolutionary relationship between archaebacteria and Gram-positive bacteria on the one hand, and Gram-negative bacteria and eukaryotes on the other. The phylogenetic analyses also indicate a polyphyletic branching of archaebacteria within the Gram-positive species. The possibility that the observed relationships are due to horizontal gene transfers can be excluded on the basis of sequence characteristics of different groups of homologs.Conclusion Our results do not support the view that archaebacteria constitute a monophyletic domain, but instead suggest a close evolutionary linkage between archaebacteria and Gram-positive bacteria. Furthermore, in contrast to the presently accepted view, eukaryotic hsp70s show a close and specific relationship to those from Gram-negative species. To explain the phylogenies based on different gene sequences, a chimeric model for the origin of the eukaryotic cell nucleus involving fusion between an archaebacterium and a Gram-negative eubacterium is proposed. Several predictions from the chimeric model are discussed.     

Cell-bound cholinesterase inTrichoderma harzianum

Cell-bound cholinesterase enzyme activity is reported for the first time in the mycelium ofTrichoderma harzianum. This enzyme hydrolyzes both the acetylcholine and the butyryl thiocholine esters. TheK _m andV _max for choline ester are 0.69 mM and 1.0 nmol acid released min^–1 g^–1 protein. However, the thiocholine ester has aK _m value of 2.2 mM andV _max value of 3.33 nmol product formed min.^–1 g^–1 protein. The enzyme is inhibited by eserine, a true classical cholinesterase inhibitor.     

Multinuclear NMR studies of intracellular cations in the prehypertensive rat kidney

We previously reported a significant derangement of intracellular free calcium ion concentration in the isolated perfused kidney of adult spontaneously hypertensive rat (SHR) (J. Biol. Chem. 267, 3637–3643, 1992). In order to investigate whether an abnormality in intracellular free calcium or another ion precedes the development of elevated blood pressure in SHR, we have now compared intracellular free Ca²⁺, Na⁺ and pH, using ³¹P, ¹⁹F, and triple quantum-filtered (TQ) ²³Na NMR, in perfused kidneys from prehypertensive young SHR and normotensive young Wistar-Kyoto (WKY) rats (5–6 weeks old) which showed no significant difference in blood pressure B.P.=120±5 mmHg and 115±3 mmHg, for SHR and WKY rats, respectively). Like the adult kidney, no significant differences in intracellular ATP concentration or intracellular pH were found between young prehypertensive SHR and normotensive WKY rat kidneys. The TQ ²³Na NMR signal was 47% higher in the SHR kidney, but, due to biological variability and measurement errors, this difference could not be shown to be statistically significant. However, a significant (40%; P<0.05) increase was found in O₂ consumption rate, a measure of the Na⁺/K⁺-ATPase activity, of the young prehypertensive SHR kidney in comparison to the age-matched WKY rat kidney (7.25±0.75 for SHR vs. 5.17±0.18 μmola O₂/min g for WKY rat, n = 6). Furthermore, a highly significant (92%; P<0.02) increase in intracellular free Ca²⁺ concentration was observed in kidneys from young SHR that had noy yet been developed high blood pressure in comparison to the kidneys from young normotensive WKY rats (648±76 nM vs. 339±39 nM, n = 4, despite the fact that there was no significant difference in blood pressure. Increased intracellular free Ca²⁺ thus appears to be part of a primary defect, in the prehypertesive young SHR kidney, which may, by way of increased release of arachidonic acid, and subsequent increased production of vasoconstricting arachidonic acid metabolites via the cytochrome P450 pathway, induce elevated blood pressure in the adult SHR.     

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands

Summary Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Adl2-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5μg/ml), hydrocortisone (0.5μg/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25μg/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca⁺² and Mg⁺²-free Hanks’, balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the ΔF508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene. Patch clamp experiments revealed that the cells expressed defective Cl⁻ channels which were not activated by Forskolin.     

Removal of endotoxin from antibody preparations for clinical use

Cell Biochemistry and Biophysics - Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is...     

Analysis of γ-Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction

Abstract: Unlike 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces consistent decreases in levels of striatal dopamine (DA) with considerably smaller and more variable effects on mouse brain levels of serotonin (5-HT) and norepinephrine (NE), a novel amine-substituted MPTP analogue, 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH₂-MPTP), administered in a standard mouse dosing paradigm for MPTP (20 mg/kg X 4) did not affect striatal DA but led to marked reductions (60–70%) in levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and NE measured in frontal cortex and hippocampus 1 week after treatment. Another 2'-substituted MPTP analogue, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine, affected cortical and hippocampal 5-HT, 5-HIAA, and NE only minimally, while markedly reducing the DA content in striatum (90%), thus indicating that the substituent (-NH₂ versus -CH₃) at the 2'position is important for the differential effects of these MPTP analogues. In a replication study with a 3-week end point, hippocampal and cortical 5-HT, 5-HIAA, and NE levels remained depressed with no indication of recovery. These results suggest that 2'-NH₂-MPTP may be a novel, regionally selective neurotoxin for serotonergic and norad-renergic nerve terminals.     

Influence of A1 cytoplasmic substitution on the downy-mildew incidence of pearl millet

Large-scale cultivation of pearl millet [Pennisetum glaucum (L.) R. Br. F₁ hybrids in India has led to increased incidence of downy-mildew (Sclerospora graminicola). There is concern that the A₁ male-sterile cytoplasm used in all the hybrids released so far is responsible for this increase. The influence of A₁ malesterile cytoplasm on downy-mildew incidence in pearl millet was studied by comparing the disease reaction of 40 pairs of F₁ hybrids, each pair carrying respectively a₁ male-sterile and normal B cytoplasm. Mean downy-mildew incidence was similar in the hybrids carrying either A₁ male-sterile or B cytoplasm. The general combining ability of lines with and without A₁ cytoplasm was found to be similar for downy-mildew incidence. These results indicated that in pearl millet A₁ cytoplasm is not associated with increased downymildew incidence. The possible danger of using only one source of cytoplasm has been briefly discussed.     

Experimental candidiasis in Japanese quail: Pathological changes

Candidiasis was experimentally produced in young Japanese quail by oral administration ofCandida albicans cells. Lesions were confined to upper digestive tract with most characteristic changes occurring on the mucosa of crop. No lesions were observed in other tissues of the body. The initial changes in the crop were characterized by thickening and yellowish-white necrotic plaques on the mucosa. From 10th day onwards, there was marked thickening and corrugations of the crop mucosa giving it a typical turkish towel appearance. Varying degree of mucosal swelling was also observed in the oesophagus and proventriculus. Two of the infected birds also revealed yellowish-white necrotic plaques on the tongue at 7th and 10th day post-infection. The prominent microscopic lesions in the crop and tongue consisted of hyperkeratosis and parakeratosis with congestion of the subepithelial tissues. Varying degree of parakeratosis and epithelial hyperplasia coupled with subepithelial oedema and hypertrophy of glands was observed in the oesophagus. The proventriculus and small intestine revealed congestion, oedema, mild to marked goblet cell hyperplasia and focal epithelial sloughing. Fungal elements could be demonstrated in the sections of tongue upto 10 days while in crop upto 14 days post-infection. Reisolation of the fungus was consistently achieved from the crop of infected birds throughout the duration of the experiment.     

Cell cycle regulation of the c-Myc transcriptional activation domain.

The product of the c-myc gene (c-Myc) is a sequence-specific DNA-binding protein that has previously been demonstrated to be required for cell cycle progression. Here we report that the c-Myc DNA binding site confers cell cycle regulation to a reporter gene in Chinese hamster ovary cells. The observed transactivation was biphasic with a small increase in G1 and a marked increase during the S-to-G2/M transition of the cell cycle. This cell cycle regulation of transactivation potential is accounted for, in part, by regulatory phosphorylation of the c-Myc transactivation domain. Together, these data demonstrate that c-Myc may have an important role in the progression of cells through both the G1 and G2 phases of the cell cycle.