Intrinsic innervation of the uterus in guinea pig and rat

The pattern of distribution of cholinergic and adrenergic nerves in the uterus of albino rats and guinea pigs was examined histochemically. In the albino rat, the uterus was found well-innervated by both adrenergic and cholinergic nerves with a clear regional variation. Dense innervation was demonstrated at the tubal and cervical ends of the uterus and in the cervix. Cholinergic nerves supplying the glands were more numerous than the adrenergic nerves which were relatively few. In the guinea-pigs, the uterus was richly innervated by adrenergic nerves with a clear regional variation. No cholinesterase-positive nerves or nerve cells were demonstrated.     

Yellow Fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functions

In flaviviruses it has been proposed that there is a coupling between genome replication and virion assembly and that nonstructural proteins are involved in this process. It was previously reported that mutations in yellow fever virus (YFV) nonstructural protein NS2A blocked production of infectious virus and that this block could be released by a suppressor mutation in NS3. Here, based on studies using a YFV replicon-based trans-packaging system as well as full-length YFV cDNA, we report that mutation of a conserved tryptophan at position 349 in the helicase domain of NS3 blocks production of infectious virus particles, revealing an as-yet-unknown role for NS3 in virus assembly. Mutation of tryptophan 349 to alanine (W349A) had no effect on viral replication, as demonstrated by wild-type levels of viral RNA amplification and protein expression in W349A-transfected cells. Although release of infectious virus was not detected, release of capsidless subviral particles was not blocked. The assembly defect in W349A could be trans-complemented inefficiently using BHK-REP cells (a cell line containing persistently replicating YFV replicon RNA). trans-complementation was also demonstrated by supplying wild-type NS2B-3 or NS3 protein alone as well as by supplying inactive NS2B-3 protein, indicating that this function of NS3 in virus assembly was independent of its known enzymatic functions.     

Anaerobic bioformation of 4-hydroxybenzoate from 4-chlorobenzoate by the coryneform bacterium NTB-1

Summary Resting cells of the coryneform strain NTB-1, previously incorrectly classified as Alcaligenes denitrificans NTB-1, quantitatively converted 4-chlorobenzoate (4-CBA) to 4-hydroxybenzoate (4-HBA) under strict anaerobic conditions in the presence of ferricyanide or nitrate. 4-HBA formation was enhanced by supplying anaerobic cells with glucose as an energy source. Using permeabilized cells it was shown that energy is not needed to drive the energy-dependent uptake of 4-CBA but also to convert 4-CBA into 4-HBA. In extracts it was subsequently demonstrated that a coenzymeA-thioester of 4-CBA is involved in the metabolism of 4-CBA. Offprint requests to: P. E. J. Groenewegen     

Octyl-beta-D-glucopyranoside extracts polyomavirus receptor moieties from the surfaces of mouse kidney cells.

Polyomavirus receptor moieties were extracted from the surfaces of mouse kidney cells with the nonionic detergent octyl-beta-D-glucopyranoside. Following extraction with this detergent, mouse kidney cells were refractory to polyomavirus infection. Binding studies demonstrated that this loss of susceptibility resulted from extraction of a peripheral membrane protein or proteins required for proper virus attachment to and infection of mouse kidney cells. Infection of extracted mouse kidney cells returned following a 2-h recovery period. However, the presence of cycloheximide or tunicamycin in the recovery media interfered with recovery from infection. Cells could be infected immediately after extraction by supplying them with the extracted moieties prior to or concomitant with infection. A complex of polyomavirus and the extracted receptor protein was formed by in vitro incubation and was stable in sucrose gradient analysis. Functional receptor moieties were prepared in the form of liposomes from the detergent extract. The virus-receptor complex was immunoprecipitated with anti-polyomavirus immunoglobulin G, and the portion of the complex contributed by the cell was identified. Immunoblot analysis of the mouse kidney cell detergent extract with a receptor-specific 125I-labeled anti-idiotypic antibody or 125I-labeled polyomavirus demonstrated several reactive proteins. Attachment of polyomavirus to mouse kidney cells, followed by extraction of the virus-receptor complex, identified polyomavirus-binding proteins similar to those observed in in vitro binding. Proteins with molecular weights of approximately 95,000, 50,000 and 25,000 to 30,000 were consistently observed in all receptor assays. The relationship between these proteins and their possible involvement as the cell receptor for polyomavirus are discussed.     

Controversies of aromatase localization in human breast cancer--stromal versus parenchymal cells

Aromatase is a key enzyme of estrogen production through conversion from serum androgens in estrogen-dependent postmenopausal breast cancer. Aromatase has been reported to be predominantly located in intratumoral stromal cells and adipocytes but not in parenchymal or carcinoma cells in breast cancer tissue. It is, however, true that there have been controversies regarding intratumoral localization of aromatase in human breast carcinoma, especially whether intratumoral production of estrogens through aromatase occurs in parenchymal or stromal cells. Results of several studies suggested that aromatase present in parenchymal carcinoma cells plays more important roles in the growth and invasion of breast carcinomas than that in stromal cells through providing higher levels of estrogens to carcinoma cells. Aromatase inhibitors are increasingly being used in place of tamoxifen after results of various clinical trials demonstrated that aromatase inhibitors are more effective in increasing survival and recurrence of estrogen-dependent breast cancer patients. Therefore, it is important to clarify the estrogen supplying pathway by aromatase inside of breast carcinoma tissues in order to evaluate the possible efficacy of aromatase inhibitor treatment. In this review, the controversies regarding these intratumoral localization patterns in human breast carcinoma will be briefly summarized.     

Stimulation of sugar loading into sieve elements of willow by potassium and sodium salts

Potassium as the chloride, nitrate or sulphate or sodium as the chloride, were applied at a concentration of 50 mM either to the xylem of stem segments or to the cambial surface of bark strips of willow. Potassium chloride increased the concentration of sucrose in sieve tube exudate collected via severed aphid stylets, without significantly affecting the volume flow rate, or the concentration of potassium in the exudate. The increase in the sucrose level in the sieve tube sap was shown to be due to a stimulation of loading, rather than to an enhancement of longitudinal transport. Potassium nitrate and sulphate or sodium chloride, were not as effective as potassium chloride in stimulating the loading of sucrose. It is suggested that uptake of the cation into cells supplying sugars to the sieve tube is linked to the rate of release of sugars by the supplying cells.     

Oxalomalate, a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase, regulates heat shock-induced apoptosis

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. The protective role of ICDH against heat shock-induced apoptosis in U937 cells was investigated in the control and the cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to heat shock, there was a distinct difference between the control cells and the cells pre-treated with 3mM oxalomalate for 3h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. The oxalomalate pre-treated cells showed significant enhancement of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to the control cells upon exposure to heat shock. This study indicates that ICDH may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.     

Substance P-like immunoreactive trigeminal ganglion cells supplying the cornea

Trigeminal ganglion cells supplying the cornea were traced with intra-axonally transported horseradish peroxidase and, subsequently studied for the presence of substance P-like immunoreactivity. Approximately 0%-30% of trigeminal ganglion cells contained immunoreactive substance P. These cells were of a small size (15-50 micrometers in diameter) and were distributed throughout the ganglion. The ganglion cells supplying the cornea were of a relatively small size as well but were confined to the anteromedial part of the ganglion. Some of these cells were found to contain immunoreactive substance P.     

Combination stem cell therapy using dental pulp stem cells and human umbilical vein endothelial cells for critical hindlimb ischemia

Narrowing of arteries supplying blood to the limbs provokes critical hindlimb ischemia (CLI). Although CLI results in irreversible sequelae, such as amputation, few therapeutic options induce the formation of new functional blood vessels. Based on the proangiogenic potentials of stem cells, in this study, it was examined whether a combination of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) could result in enhanced therapeutic effects of stem cells for CLI compared with those of DPSCs or HUVECs alone. The DPSCs+ HUVECs combination therapy resulted in significantly higher blood flow and lower ischemia damage than DPSCs or HUVECs alone. The improved therapeutic effects in the DPSCs+ HUVECs group were accompanied by a significantly higher number of microvessels in the ischemic tissue than in the other groups. In vitro proliferation and tube formation assay showed that VEGF in the conditioned media of DPSCs induced proliferation and vessel-like tube formation of HUVECs. Altogether, our results demonstrated that the combination of DPSCs and HUVECs had significantly better therapeutic effects on CLI via VEGF-mediated crosstalk. This combinational strategy could be used to develop novel clinical protocols for CLI proangiogenic regenerative treatments.     

OXYGEN CONCENTRATION WITHIN THE NITROGEN-FIXING ROOT NODULES OF MYRICA GALE L.

Microelectrodes were used to study the oxygen concentration within Myrica gale L. nodules. Low oxygen concentrations were found only in the region of the mature, nitrogen-fixing endophyte, and appeared to correspond to clusters of infected host cells. The oxygen concentration in the remainder of the nodule was much higher. Interconnected intercellular air spaces were demonstrated by infiltration with India ink. Infiltration of the spaces with water greatly reduced oxygen concentration throughout the nodule, indicating that they function in supplying oxygen to the infected cells and remainder of the nodule. These results differ from those found previously for soybean nodules and provide evidence that legume and actinorhizal nodules have different mechanisms for protecting nitrogenase from oxygen.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Anti-proliferative activity of L-651,582 correlates with calcium-mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1, 2,3-triazole-4-carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H-hypoxanthine, 14C-adenine, and 14C-formate incorporation into nucleotide pools in Madin-Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L-651,582 inhibited incorporation of 3H-hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose-5-phosphate. PRPP synthetase was not inhibited directly by the compound, indicating regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.     

Response to endothelin-1 in arteries from human colorectal tumours: role of endothelin receptors

To examine the reaction of tumour arteries to endothelin-1, we obtained arteries supplying blood flow to colorectal tumours from patients, as well as mesenteric arteries supplying the normal colon tissue from the same patients and mesenteric arteries from patients without a colorectal tumour pathology. The contraction in response to endothelin-1 and the relaxation produced by bradykinin was recorded in each of these arteries. Accordingly, the sensitivity to endothelin-1 but not the maximal response, was higher in the arteries supplying colorectal tumours than in mesenteric arteries supplying normal colon or in mesenteric arteries from patients with no tumour pathology. The contraction produced by endothelin-1 was not modified by exposure to L-NAME or meclofenamate in arteries supplying both the tumour and the normal colon. The endothelin ET(A) andET(B) receptors were expressed similarly in arteries supplying the tumour or normal colon. However, the antagonist of the endothelin ET(B) receptors BQ788 (10(-6) M) decreased the contractions in the arteries supplying the tumour but not in those supplying the normal colon. By contrast, the antagonist of endothelin ET(A) receptors BQ123 (10(-6) M) reduced the contraction equally in both these types of arteries. Likewise, in arteries precontracted with U46619, the relaxation in response to bradykinin was similar in all three types of arteries. Together, these results suggest that the arteries supplying human colorectal tumours are more sensitive to endothelin-1, which could be due to the enhanced activity of endothelin ET(B) receptors in the absence of any change in the modulatory effect of nitric oxide or prostanoids in the arterial response to this peptide.     

Trimethoprim induces heat shock proteins and protein aggregation in E. coli cells

Trimethoprim (TMP), an inhibitor of dihydrofolate reductase, decreases the level of tetrahydrofolate supplying one-carbon units for biosynthesis of nucleotides, proteins, and panthotenate. We have demonstrated for the first time that one of the effects of the TMP action in E. coli cells is protein aggregation and induction of heat shock proteins (Hsps). TMP caused induction of DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps. Among these Hsps, IbpA/B were most efficiently induced by TMP and coaggregated with the insoluble proteins. Upon folate stress, deletion of the delta ibpA/B operon resulted in increased protein aggregation but did not influence cell viability.     

Protein kinase C-dependent supply of secretory granules to the plasma membrane

To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system.     

Nitrogen anabolism underlies the importance of glutaminolysis in proliferating cells

Warburg effect and glutaminolysis are the two most noticeable metabolic features of tumor cells. A recent hypothesis is that tumor cells use glutamine as a preferred carbon source for energy and reducing power, which has been used to explain both Warburg effect and glutaminolysis. Here we provide evidence to demonstrate that supplying nitrogen, not the carbon skeleton, underlies the biological importance of glutaminolysis for proliferating cells. We show alternative nitrogen supplying mechanisms rescue Hep3B and HeLa cell proliferation in glutamine-free media. We conclude that cellular demand for nitrogen is a driving force of glutaminolysis for proliferating cells, and propose that glutamine addiction and Warburg effect are metabolic consequences of coordinated utilization of nitrogen and carbon sources for biosynthesis.     

Neurobiological mechanisms of the meridian and the propagation of needle feeling along the meridian pathway

The present experiments attempt to find the meridian phenomenon and how the needle feeling propagates along the given meridian channels. The neurobiological mechanisms of the meridian were studied with neuroelectrical recording from the motor neurons and CB-HRP retrograde histochemistry technique in both rats and cats. The results demonstrated that most, but not all, of alpha motor neurons supplying a muscle group of a given meridian were selectively activated by afferent inputs originating not only from homonymous or heterogeneous, but synergistic muscle, but also from the skin nerve overlying the muscle group of the homonymous meridian. However, the afferent inputs from the heterogeneous meridian have very weak or no effect. On the other hand, the labeled motor neurons supplying a given meridian muscles form a discrete longitudinal column with a definite bound in the lateral ventral horn. There are oriented dendro-dendristes projections between the labeled motor neurons.The characteristics of both sel