Different roles of proteolipids and 70-kDa subunits of V-ATPase in growth and death of cultured human cells

BACKGROUND: The vacuolar-type proton-translocating adenosine triphosphatase (V-ATPase) plays important roles in cell growth and tumour progression. V-ATPase is composed of two distinct structures, a hydrophilic catalytic cytosolic sector (V(1)) and a hydrophobic transmembrane sector (V(0)). The V(1) sector is composed of 5-8 different subunits with the structure A(3)B(3)C(1)D(1)E(1)F(1)G(1)H(1). The V0 sector is composed of 5 different subunits with the structure 1161381191166. The over-expression of 16-kDa proteolipid subunit of V-ATPase in the perinuclear region of the human adventitial fibroblasts promotes phenotypic modulation that contributes to neointimal formation and medial thickening. A relationship between oncogenicity and the expression of the 16-kDa proteolipid has also been suggested in human pancreatic carcinoma tissue. RESULTS: We found that the mRNA levels of the 16-kDa proteolipid but not of the 70-kDa subunit of V-ATPase in human myofibroblasts were more abundant in serum-containing medium (MF(+) cells) than serum-free medium (MF(-) cells). In HeLa cells, the levels of mRNA and protein of the 16-kDa, 21-kDa or 70-kDa were clearly suppressed when the corresponding anti-sense oligonucleotides were administered to the culture medium. The growth rate and viability (mostly due to necrosis) of HeLa cells were reduced markedly by the 16-kDa and 21-kDa anti-sense, but little by the 70-kDa anti-sense, and not at all by any sense oligonucleotides. The localization of 16-kDa/21-kDa proteolipid subunits was different from that of the 70-kDa subunit in HeLa cells. CONCLUSION: These results suggest that the 16-kDa and 21-kDa proteolipid subunits of the V0 sector play crucial roles in growth and death of cultured human cells. Our results may provide new insights into the mechanism and therapeutic implications for vessel wall hyperplasia and tumorigenesis.     

Dominant negative protein kinase Cbeta improves 1alpha, 25-dihydroxy vitamin D3-induced insulin resistance

1alpha,25-Dihydroxy vitamin D3 (1,25D3) activates conventional PKC and may subsequently lead to insulin resistance. Previous studies from our laboratory have shown that pretreatment with 10 nM-10 microM 1,25D3 dose-responsively suppressed insulin-induced glucose. To assess PKC(beta)-mediated inhibition of insulin-induced glucose uptake in rat adipocytes, we preincubated with Go6976 and LY379196, conventional PKC inhibitors, and found they abolished the 1,25D3-mediated inhibitory effect on insulin-induced 2-deoxyglucose (DOG) uptake. Moreover, the inhibitory effect of 1,25D3 on insulin-induced DOG uptake was abrogated in adipocytes overexpressed with dominant negative PKC(beta), but not in those overexpressed with wild type PKC(beta). These results suggest that 1,25D3 reduces insulin-induced glucose uptake via activation of PKC(beta) in rat adipocytes.     

The effect of IL4 +33C/T polymorphism on risk of Japanese sporadic Alzheimer's disease

Interleukin-4 (IL4) is an anti-inflammatory cytokine that may play a role in the inflammation pathology observed surrounding senile plaques, and may also associate with Alzheimer's disease (AD) pathogenesis. Recently, it has been reported that a single nucleotide polymorphism in the IL4 gene promoter region, IL4 +33C/T polymorphism, associates with its phenotype. It was thought that the IL4 +33C/T polymorphism causing high IL4 production may reduce the risk for AD. In the present study, therefore, we investigated this mutation in 108 healthy controls and in 178 sporadic AD cases by the polymerase chain reaction restriction fragment length polymorphism method in a Japanese AD population. Allelic frequencies with +33C/T polymorphism in the gene were 35.6 and 32.6% in the control and AD groups, respectively. Our results failed to demonstrate an association between this polymorphism and Japanese sporadic AD. We also tested whether the IL4 functional variants were regulated by this polymorphism in a portion of the subjects (16 AD cases and 13 control cases). We could not find any relationship between the IL4 +33C/T polymorphism and plasma IL4 concentration.     

Topographical relationships between the brainstem auditory and somatosensory evoked potentials and the location of lesions in posterior fossa stroke

The topographical relationships between the location of brainstem lesions detected by magnetic resonance imaging and abnormality of brainstem auditory evoked potentials (BAEPs) and short-latency somatosensory evoked potentials (SSEPs) were studied in 57 patients with stroke in the posterior fossa. Abnormal BAEPs or SSEPs were associated with lesions involving the pontine tegmentum, and abnormal BAEPs also with lesions at the cerebellar peduncle. Absence of the V wave in BAEPs and N20 in SSEPs was associated with a localized overlapping area in the pontine tegmentum contralateral to stimulation. The overlapping area associated with loss of N20 coincided with the location of the medial lemniscus. Lesions widely involving the pontine tegmentum caused the disappearance of multiple waves in the BAEPs and SSEPs. Patients who entered prolonged coma or died had total loss of the III, IV, and V waves, bilateral absence to the contralateral response in BAEPs, or loss of N18 in SSEPs. The loss of N18 in SSEPs had a statistically significant correlation with bad outcome, which suggests the superiority of SSEPs for predicting the outcome of stroke and indicates the involvement of some system excluding the medial lemniscus in the generation of N18.     

Reference birth weight, length, chest circumference, and head circumference by gestational age in Japanese twins

BACKGROUND: Numerous birth weight standards for twins have been reported in western countries, whereas little is in Japan. The aim of this study is to present birth weight, birth length, chest circumference, and head circumference references, clarifying features related to these body size parameters, and to compare our birth weight references with recent report of birth weight norms of Japanese twins using the vital statistics. METHODS: The subjects consisted of 1,061 twin pairs in total, with birth years ranging from 1968 through 1990. Data was obtained from the Twins Protocol Questionnaire, which asked for information about twins' growth and development in infancy, and the "Maternal and Child Health Handbook," which was presented by Ministry of Health and Welfare. Statistical means, standard deviations, and selected percentiles by gestational age were calculated and smoothed using data that contained at least gestational age and one of the four items. RESULTS: Birth weight was significantly lighter than that of singletons when three additional parameters, especially chest and head circumference, were not measured. Gestational age was correlated with weight, length, chest circumference, and head circumference, in that order, for both sexes. Compared with singletons, birth weight difference in twins was marked and slight difference was observed as to length, whereas no difference was observed as to chest and head circumference. The present results as to birth weight were consistently similar to the birth weight norms of twins using vital statistics in Japan. CONCLUSION: Growth standards for twins, especially as to birth weight, are essential to understand and evaluate intrauterine growth of twins.     

Ingestion of an indigestible saccharide, difructose anhydride III, partially prevents the tannic acid-induced suppression of iron absorption in rats

Dietary tannic acid (TA) inhibits iron absorption and some indigestible oligosaccharides have been shown to promote mineral absorption. In this study, we examined whether difructose anhydride III (DFA III) or fructooligosaccharide (FOS) stimulate iron absorption in TA-fed rats. Two experiments were conducted using male Sprague-Dawley rats (weighing 90-110 g) in a randomized block design. Rats were fed control, DFA III or FOS (30 g/kg) diets in expt. 1, and control, TAcontrol, TAFOS or TADFA III (TA, 15 g/kg) diets in expt. 2 for 3 wk during which blood sampling was performed weekly and fecal collection twice. In expt. 1, apparent iron absorption was higher (P < 0.001) in the DFA III-fed (65.7 and 55.9%, d 8-10 and 19-21) and FOS-fed (59.9%, d 19-21) groups than in the control group (48.4 and 45.4%, d 8-10 and 19-21) without differences in blood hemoglobin concentrations or hematocrits. TA feeding reduced hemoglobin concentrations and hematocrits (119.1 g/L, 0.360; P < 0.001), and the feeding of TADFA III partially improved this anemic condition (129.6 g/L, 0.403), whereas TAFOS feeding did not influence these variables (120.6 g/L, 0.342; expt. 2). Iron absorption was lower in the TA-fed groups (19.8%; P < 0.001) than in the control group (49.4%), whereas the absorption in both TA-fed indigestible sugar groups was higher (DFA III, 43.2 and 38.2%, d 8-10 and 19-21; FOS, 39.4%, d 8-10; P < 0.001) than in the TA-control group except for the TAFOS-fed group (25.1%, d 19-21). Serum iron concentrations, unsaturated iron-binding capacities, total iron-binding capacities and transferrin saturations (%) were not improved by the feeding of TADFA III or TAFOS. Furthermore, liver iron concentrations were decreased by TA feeding (P < 0.001) and were not increased by the feeding of indigestible sugars. The feeding of DFA III or FOS decreased the pH of the cecal contents (P < 0.001) while increasing major organic acid pools. In all groups fed TA, approximately 18% of the ingested TA was recovered in the feces. Our results demonstrate that TA reduces iron absorption and induces anemia, conditions that are partially prevented by the feeding of DFA III, but not FOS.     

A case of partial carnitine palmitoyltransferase deficiency in a patient undergoing chronic hemodialysis

A 71-year-old male undergoing hemodialysis for chronic renal failure presented with proximal muscle weakness. He had normal levels of serum creatine phosphokinase. The results of nerve conduction velocity studies and a needle-exploration electromyogram were normal. Ultrasonography revealed adenomatous enlargement of the parathyroid glands, and he had marked elevation of the serum parathormone level. The level of serum free carnitine before hemodialysis was significantly lower than normal, while the acyl/free ratio was high. However, the muscle carnitine content was within the normal range. Interestingly, partial inactivation of carnitine palmitoyltransferase activity in the muscle was observed in association with the elevation of the serum parathormone level. Uremic myopathy in this case may be caused not only by abnormal carnitine metabolism but could also be attributable to the partial carnitine palmitoyltransferase deficiency associated with secondary hyperparathyroidism.     

Different molecular mechanisms of vitamin D(3) receptor antagonists

Two structurally different antagonists of the nuclear hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the 25-carboxylic ester ZK159222 and the 26,23-lactone TEI-9647, have recently been described. In this study, the molecular mechanisms and the efficacy of both antagonists were compared. ZK159222 showed similar potency and sensitivity to 1alpha,25(OH)(2)D(3) in ligand-dependent gel shift assays using the vitamin D receptor (VDR), the retinoid X receptor, and specific DNA binding sites, whereas TEI-9647 displayed reduced potency and >10-fold lower sensitivity in this assay system. Limited protease digestion and gel shift clipping assays showed that the two antagonists stabilized individual patterns of VDR conformations. Both antagonists prevented the interaction of the VDR with coactivator proteins, as demonstrated by GST-pull-down and supershift assays; like the natural hormone, however, they were able to induce a dissociation of corepressor proteins. Interestingly, ZK159222 demonstrated functional antagonism in reporter gene assays both in HeLa and MCF-7 cells, whereas TEI-9647 functioned as a less sensitive antagonist only in MCF-7 cells. In conclusion, the two 1alpha,25(OH)(2)D(3) analogs act in part via different molecular mechanisms, which allows us to speculate that ZK159222 is a more complete antagonist and TEI-9647 a more selective antagonist.     

Enhancement of antiproliferative effects of interleukin-1beta and tumor necrosis factor-alpha on human prostate cancer LNCaP cells by coculture with normal fibroblasts through secreted interleukin-6.

The cell-cell interactions between tumor cells and stromal cells are considered to be important in the regulation of tumor development at primary and metastatic secondary sites. We studied the effects of various cytokines on the cell-cell interactions between androgen-dependent LNCaP or androgen-independent PC-3 human prostate cancer cell lines and normal fibroblasts using a co-culture system. Among the tested combinations of cytokines and fibroblasts, strong modulations of cytokine actions were seen in coculture with human normal fibroblasts WI-38. While interleukin (IL)-1beta or tumor necrosis factor-alpha (TNF-alpha) partially suppressed LNCaP cell growth in monoculture, each cytokine completely inhibited it in the case of coculture with WI-38 cells. On the other hand, they did not inhibit PC-3 cell growth significantly, regardless of monoculture or coculture. Conditioned medium prepared from WI-38 cells pretreated with IL-1beta or TNF-alpha also strongly inhibited LNCaP cell growth. In the conditioned medium, marked IL-6 secretion was induced from WI-38 cells by IL-1beta or TNF-alpha. Furthermore, neutralizing antibodies to IL-6 or IL-6 receptor abrogated the antiproliferative effects of IL-1beta- and TNF-alpha-pretreated WI-38 conditioned medium. These results demonstrate that the antiproliferative effects of IL-1beta and TNF-alpha on prostate cancer cells are enhanced by coculture with normal fibroblasts through some diffusible factor(s), such as IL-6, from the stimulated fibroblasts.