Effects of niacin deficiency on the levels of soluble proteins and enzyme activities in various tissues of Japanese quail

The effects of niacin deficiency on the levels of soluble proteins and enzyme activities of Japanese quail have been investigated. SDS-polyacrylamide gel electrophoresis revealed that in the pectoral muscle the soluble proteins with molecular masses corresponding to 181, 128, 93, 76, 72, 62, 56, 43, 41, 28 and 20 kDa were present in lower amounts but those of 60, 50 and 37 kDa were present in higher amounts. In the heart the soluble proteins with a molecular mass of 181 kDa were present in lower amounts and in the brain those of 43 kDa were present in lower amounts but those of 221 kDa were present in higher amounts. In the intestine the soluble proteins with molecular masses corresponding to 181, 102, 83, 74, 72, 44 and 40 kDa were present in lower amounts but those of 41 kDa and 18 kDa were present in higher amounts. There was a marked reduction in the level of NAD and NADPH in the pectoral muscle of niacin deficient quail but not in other tissues. The specific activity of glyceraldehyde-3-phosphate dehydrogenase decreased markedly both in the liver and pectoral muscle of niacin deficient quail whereas that of 6-phosphogluconate dehydrogenase and malic enzyme decreased markedly in the liver or pectoral muscle, respectively. In contrast, the specific activity of acetylcholinesterase and carboxypeptidase increased markedly in the liver or the pectoral muscle, respectively. The results suggest that a severe niacin deficiency exerted specific effects on levels of some soluble proteins particularly in the pectoral muscle and intestine and on activities of certain enzymes in the liver and the pectoral muscle.     

Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites

Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX4GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites.     

Gi-mediated stimulation of type II adenylyl cyclase is augmented by Gq-coupled receptor activation and phorbol ester treatment

Synergism between Gs- and Gi- or Gq-dependent signaling pathways has been demonstrated in the stimulation of type II adenylyl cyclase (AC-II). Provision of activated alpha s is known to allow numerous Gi-coupled receptors to stimulate AC-II and to potentiate the responses to Gq-coupled receptors. To explore possible interactions between Gi- and Gq-coupled receptors that are independent of alpha s, the activity of AC-II was determined after the activation of Gi- and Gq-regulated pathways. Human embryonic kidney 293 cells were transiently cotransfected with cDNAs encoding AC-II and various G-protein-coupled receptors. Agonist-bound Gi-coupled receptors (including the formyl peptide, dopamine-D2, and delta-opioid receptors) stimulated AC-II activity in the absence of activated alpha s, provided that the cells were treated with 100 nM phorbol 12-myristate 13-acetate. Activation of protein kinase C (PKC) thus appears to relieve the requirement for the presence of activated alpha s. Stimulation of PKC via Gq-coupled receptors also allowed Gi-coupled receptors to activate AC-II. Coexpression of the m1 muscarinic receptor with the dopamine-D2 receptor permitted dopamine to stimulate AC-II in the presence of carbachol. The phorbol ester-permissive and alpha s-independent stimulation was mediated by G-protein beta gamma subunits because it was blocked by the beta gamma scavengers alpha t and beta-adrenergic receptor kinase. These results show that AC-II can efficiently integrate signals generated by Gq- and Gi-coupled receptors via a mechanism that is independent of Gs.     

Anabolic effect of genistein and genistin on bone metabolism in the femoral-metaphyseal tissues of elderly rats: the genistein effect is enhanced by zinc

The effect of genistein and genistin on bone components in the femoral-metaphyseal tissues obtained from elderly female rats was investigated in vitro. The metaphyseal tissues were cultured for 24 h in a medium containing either vehicle, genistein (10(-8)-10(-5) M) or genistin (10(-7)-10(-5) M). The presence of genistein or genistin caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the metaphyseal tissues. The effect of genistein was greater than that of genistin. The bone components increased by genistein (10(-5) M) or genistin (10(-5) M) were completely blocked by the presence of cycloheximide (10(-6) M). The presence of zinc sulfate (10(-5) M) caused a significant increase in the genistein (10(-5) M)-elevated alkaline phosphatase activity, DNA and calcium contents. The enhancement with zinc was not seen by genistin (10(-5) M). The stimulatory effect of zinc on the genistein-induced increase in bone components of the metaphyseal tissues was completely blocked by the presence of cycloheximide (10(-6) M). The present results suggest that genistein and genistin have an anabolic effect on bone metabolism in the femoral-metaphyseal tissues of elderly rats, and that the genistein effect is enhanced by zinc, an essential trace element.     

Islets of Langerhans generate wavelike electric activity modulated by glucose concentration

The electric activity of whole islets of Langerhans was monitored for the first time in this study. Measurements were made from single islets isolated from mice, hamsters, gerbils, and rats by means of external electrodes. Well-structured synchronized potential spikes up to 0.5 mV in amplitude with a stable frequency of 0.5-2 Hz were measured. Spike generation had a glucose concentration threshold. In the physiological range of each animal species, firing rate was an approximate linear function of glucose concentration. At low glucose concentrations, firing became intermittent, i.e., in bursts, while in the physiological range and above, firing was typically continuous. Simultaneous measurements from two locations on an islet indicate that the measured activity reflects the propagation of an excitation wave throughout the islet. This, together with signal synchronization, suggests that the islets contain a functional pacemaker (FPM) from which excitation propagates by means of gap junctions to the rest of the islet cells (mostly beta-cells). Thus, the electric characteristics of the individual beta-cells are functionally masked so that the islet acts as a single functional unit. In view of the dependency of insulin secretion on the islet's electric activity, the islet glucose-insulin dose-response characteristics must be determined by those of the FPM.     

Lymph node involvement and tumor depth in rectal cancers: an analysis of 805 patients

BACKGROUND: Superficial rectal tumors are said to involve regional lymph nodes rarely. This presumption must be proven beyond any doubt if less radical surgery is to be offered for such patients. PATIENTS AND METHODS: Eight hundred five cases (467 males; median age, 64 (range, 19-97) years) of rectal cancer were reviewed. RESULTS: Lymph node positivity, number of lymph nodes involved, lymphatic vessel, and venous and perineural invasion were significantly increased with increasing depth of invasion of tumor through the bowel wall in univariate analysis. The percentage of lymph node involvement at each tumor depth was as follows: T1, 5.7 percent; T2, 19.6 percent; T3, 65.7 percent; T4, 78.8 percent. Overall lymph node involvement was 59 percent. For patients younger than 45 years of age, the percentage of lymph node involvement was 33.3, 30, 69.3, and 83.3 percent compared with 3.1, 8.4, 64.2, and 78.8 percent for patients aged 45 years or above for T1, T2, T3, and T4, respectively. CONCLUSION: Increased depths of tumor penetration beyond T1 and age less than 45 years have an excessive incidence of lymph node positivity. The finding of lymphatic vessel invasion on biopsy is highly indicative of lymph node metastasis.     

Mass loss in urethane/TEGDMA- and Bis-GMA/TEGDMA-based resin composites during post-cure heating

OBJECTIVES: This research examined weight loss of commercial UDMA/TEGDMA- and Bis-GMA/TEGDMA-based resin composites at a variety of post-cure temperatures. Weight loss profiles of individual monomer components were also tested at elevated temperature: Bis-GMA, ethoxylated Bis-GMA (EBis-GMA), urethane dimethacrylate (UDMA), and triethyleneglycol dimethacrylate (TEGDMA). METHODS: Disc-shaped composite specimens (1 x 5 mm, approximately 50 mg) were light-cured and then isothermally post-cure heated in a thermogravimetric analysis (TGA) unit at either 50 degrees, 75 degrees, 100 degrees, 125 degrees or 150 degrees C. A single specimen was made for each post-cure temperature for each product (a total of 10 discs). Individual monomer components were heated to 800 degrees C. Filler and organic phase weight percentages were determined by ashing cured composite in the TGA. Weight loss differences between resin systems at various post-cure temperatures were analyzed using linear regression. RESULTS: For each type of composite, loss of volatile component increased with both elevated post-cure temperature as well as duration of heat exposure. Using recommended post-cure temperature and time (125 degrees C for 7.5 minutes), there was no difference in weight loss profile between the two products: both exhibited 1.3% loss of resin component. After 10 min of heating, the Bis-GMA-based product always demonstrated a greater weight loss than the UDMA material. Weight loss could not be attributed to any specific monomer. SIGNIFICANCE: Specimen weight loss during post-cure heating may result in a depletion of leachable, unreacted material at the restoration surface, possibly enhancing material properties at that location. This decrease would also potentially reduce the biological impact of leachable materials. Loss of volatile components from post-cure heating would affect the accuracy of infrared spectroscopic techniques in determining monomer conversion values.