Type II collagen-induced autoimmune endolymphatic hydrops in guinea pig.

Endolymphatic hydrops was induced in guinea pigs by immunizing them with native bovine type II collagen. Histopathologic changes consisted of moderate extension of the Reissner's membrane, spiral ganglion degeneration, atrophied organ of Corti, and mild atrophy of the surface epithelium in the endolymphatic duct. These findings suggest that an immune response directed against type II collagen--a type of collagen found in the membranous labyrinth, subepithelial layer of the endolymphatic duct, spiral ligament, and enchondral layer of the otic capsule--may induce endolymphatic hydrops.

Gene duplication and fusion have occurred frequently in the evolution of phosphagen kinases--a two-domain arginine kinase from the clam Pseudocardium sachalinensis.

In contrast to the 40 kDa arginine kinases from Molllusca and Arthropoda, the adductor muscle of the marine clam Pseudocardium sachalinensis contains an unusual arginine kinase consisting of an 86 kDa subunit. The cDNA encoding the 86 kDa arginine kinase was amplified by PCR and the cDNA-derived amino acid sequence of 724 residues was determined. The exact molecular mass for the protein was calculated to be 80941 Da. The amino acid sequence clearly indicates that Pseudocardium arginine kinase has a two-domain structure: the first domain residues 1-363 and the second domain 364-724. The two domains, which are separated by an intron of 176 bp in the gene, show 62% amino acid sequence identity. This two-domain arginine kinase from a mollusc represents yet another multiple-domain enzyme observed in the phosphagen kinase enzyme family. Two-domain and three-domain enzymes have been observed in three other diverse invertebrate groups. Thus, it is clear that gene duplication and subsequent fusion have occurred frequently, and likely independently, during the course of the evolution of this enzyme family. Comparison of the amino acid sequence in the GS region (a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) suggests that the first domain of Pseudocardium arginine kinase might not retain a complete enzyme activity, because the Asp-7 in the GS region, which is assumed to be involved in the recognition of the positive charge of arginine, was replaced by a Gly residue in the first domain.

Schedule-dependent interaction between paclitaxel and doxorubicin in human cancer cell lines in vitro.

The schedule-dependent interaction of paclitaxel and doxorubicin was evaluated in four human cancer cell lines. The cells were exposed simultaneously or sequentially to the two agents for 24 h, and were then incubated in drug-free medium for 4 and 3 days, respectively. The cell growth inhibitions were determined by the MTT assay. The cytotoxic interactions at the IC80 level were evaluated by the isobologram method of Steel and Peckham. In non-small cell lung cancer A549, breast cancer MCF7 and colon cancer WiDr cells, antagonistic effects were observed for the paclitaxel and doxorubicin combination on simultaneous exposure to the two agents and on sequential exposure to doxorubicin followed by paclitaxel, while additive effects were observed for the combination on sequential exposure to paclitaxel followed by doxorubicin. In ovarian cancer PA1 cells, additive effects were observed for all schedules. These findings suggest that sequential administration of paclitaxel followed by doxorubicin may be the most suitable sequence, while the simultaneous administration of the two agents and the sequential administration of doxorubicin followed by paclitaxel may result in less tumour cell kill than anticipated. Further preclinical and clinical studies are required to elucidate the relationship between paclitaxel and doxorubicin with regard to both antitumour activity and toxicity.

RT-PCR analysis of mRNA expression of natriuretic peptide family and their receptors in rat inner ear.

To assess the possible physiological role of the atrial natriuretic peptide (ANP) family, we investigated the expression of mRNA of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and their receptors in rat inner ear using the reverse transcription-polymerase chain reaction method. ANP and CNP message bands were detected in the inner ear, but the BNP message band was not. Amplification products of the expected sizes of ANP-A, ANP-B and ANP-C receptors were detected in the inner ear. These results suggest that natriuretic peptide family may influence the function of the inner ear through the ANP-A, ANP-B, and ANP-C receptors.

Light chains of abalone myosin. UV absorption difference spectrum and resensitization of desensitized scallop myosin.

1. Abalone myosin consisted of one kind of heavy chain and two kinds of light chain according to SDS gel electrophoresis. The molecular weights of the light chains were estimated as 16,600 daltons (Light Chain-1: LC-1) and 14,500 daltons (Light Chain-2: LC-2). 2. The amino acid composition of LC-2 was not appreciably different from that of LC-1 except that the valine residue was 1 mol per mol of LC-2. 3. both LC-1 and LC-2 showed a calcium-induced UV absorption difference spectrum through the apparent binding constants for Ca2+ were low (2.5 x 10(-3) M for LC-1 and 3.2 x 10(-4) M for LC-2). 4. The modification of carboxyl groups of LC-2 by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide caused the disappearance of the calcium-induced UV absorption difference spectrum. 5. Manganese ion could also induce a UV absorption difference spectrum with these light chains although the concentration of Mn2+ required to produce the difference spectrum was very high. 6. Abalone LC-2 bound to desensitized scallop myosin and restored the calcium sensitivity of the myosin but LC-1 did not.

Low molecular weight components (g-chains) of myosin from rabbit skeletal muscle. Separation, amino acid compositions and contents in myosin.

Low molecular weight components (g1, g2, and g3) were isolated from rabbit skeletal muscle myosin and their amino acid compositions were analyzed. One mole tryptophan was found in g1 and in g2, but none in g3. One mole of acetic acid was found per mole of each g-chain and it was concluded that the N-terminal groups of all three g-chains are acetylated. The minimum molecular weight of the g-chains were estimated from their amino acid compositions. It was estimated by SDS-disc electrophoresis that 1 mole of myosin contained 0.90, 1.7, and 0.63 moles of g1, g2, and g3, respectively. Similar values were obtained with psoas muscle myosin, but in heavy meromyosin prepared from skeletal muscle myosin the content of g2 was much lower, and that of g3 was much higher.

Cortical and subcortical vestibular response to caloric stimulation detected by functional magnetic resonance imaging.

The posterior insula, central sulcus, and inferior parietal lobule including the intraparietal sulcus have been considered the vestibular cortex based on functional brain mapping in humans as well as experiments in lower primates. The same regions receive optokinetic, visual, and proprioceptive projections. We examined the cortical and subcortical projection of vestibular activity with visual and proprioceptive input eliminated during caloric stimulation (CS), using functional magnetic resonance imaging (fMRI). Single-shot gradient-echo echoplanar image (EPI) volumes were sensitive to BOLD contrast in oblique orientation. We adopted a pharmacokinetic model for analysis of imaging data from 10 subjects as a group. The insular gyrus, intraparietal sulcus, superior temporal gyrus, hippocampus, cingulate gyrus, and thalamus showed activation by CS. Cortical and subcortical activation during CS in the present study was observed within regions less precisely delineated by other methods. As intraparietal sulcus activation showed right hemispheric dominance, this region may have an oculomotor projection as well as the vestibular input.

Detection of C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP-B) receptor mRNAs in rat inner ear.

C-Type natriuretic peptide (CNP) is the third member of the natriuretic peptide family which plays an important role in body fluid homeostasis. To determine a possible role of CNP in regulation of an inner ear fluid, we investigated the expression of CNP and atrial natriuretic peptide B receptor (ANP-B receptor) mRNAs in rat inner ear using a reverse transcription polymerase chain reaction (RT-PCR) method. Amplification products with sizes expected for CNP and ANP-B were detected in the inner ear. After cloning and analysis, the sequences for PCR products were identical to those of CNP or ANP-B receptor in the brain. These results indicate that both CNP and ANP-B receptor are expressed in the inner ear of the rat and suggest that CNP may play a role in inner ear function (such as regulation of inner ear fluid) in an autocrine and/or paracrine manner.

Vasopressin and oxytocin receptor mRNAs are expressed in the rat inner ear.

The cause of endolymphatic hydrops, a characteristic finding in Menière's disease, is not known. To study the possible involvement of the neurohormones vasopressin and oxytocin in this condition, we investigated whether transcripts of the genes encoding the arginine vasopressin (AVP) and oxytocin receptors are expressed in the rat inner ear. Utilizing the reverse transcription-polymerase chain reaction (RT-PCR) method, primers specific for each receptor showed a single message band of the expected size in the rat inner ear. When the PCR products were cloned, the sequences were identical to those of the real-type (V2) AVP receptor and oxytocin receptor transcripts. The finding of vasopressin and oxytocin receptor mRNAs in the inner ear suggests that these neurohypophyseal hormones may have roles in the regulation of inner ear fluid. In particular, the presence of vasopressin receptor mRNA in the inner ear supports the hypothesis of a relationship between high plasma vasopressin levels and endolymphatic hydrops.

Regulation of inner ear fluid in the rat by vasopressin.

The anti-diuretic hormone vasopressin has been shown to be important in regulating inner ear fluid. The diuretic hormone, CNP, and its receptor, ANP-B receptor, may also function in the regulation of inner ear fluid. To determine whether vasopressin directly affects the fluid level, we infused this hormone to rat and assay of V2-AVP receptor mRNA by semiquantitative RT-PCR demonstrated a significantly lower level of this transcript in vasopressin-infused animals than in saline-infused animals. The levels of CNP and ANP-B receptors mRNA, however, were the same in both groups of rats. Results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps by down-regulating the number of vasopressin receptors.

In vitro schedule-dependent interaction between paclitaxel and cisplatin in human carcinoma cell lines.

The schedule-dependent interaction of paclitaxel and cisplatin was studied in four human carcinoma cell lines: non-small cell lung cancer, A549; breast cancer, MCF7; ovarian cancer, PA1; and colon cancer, WiDr cells. The cells were exposed simultaneously to the drugs for 24 h and sequentially to paclitaxel first for 24 h followed by cisplatin for 24 h, or vice versa, and then incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was then determined by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the IC80 level were analyzed by the isobologram method. On simultaneous exposure to paclitaxel and cisplatin, additive and subadditive (slight antagonistic) effects were observed in A549, MCF7, and PA1 cells, while sub-additive and protective (antagonistic) effects were observed in WiDr cells. On sequential exposure to paclitaxel first, followed by cisplatin, additive effects were observed in all cell lines. On sequential exposure to cisplatin first, followed by paclitaxel, additive effects were observed in PA1 cells, while additive, sub-additive, and protective effects were observed in A549, MCF7, and WiDr cells. These findings suggest that the interaction of paclitaxel and cisplatin is schedule- and cell line-dependent. The optimal schedule of this combination may be paclitaxel first followed by cisplatin.

Schedule-dependent interactions between paclitaxel and etoposide in human carcinoma cell lines in vitro.

Clinical studies of paclitaxel in combination with etoposide against solid tumors have been carried out. The combination schedules used in these studies are different. We studied the cytotoxic effects of paclitaxel with etoposide against four human cancer cell lines in vitro to determine the optimal schedule of this combination at the cellular level. Cells were exposed simultaneously to paclitaxel and to etoposide for 24 h or sequentially to one drug for 24 h followed by the other for 24 h, after which they were incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was determined by an MTT reduction assay. The effects of drug combinations at concentrations producing 80% inhibition (IC(80)) were analyzed by the isobologram method of Steel and Peckham. The cytotoxic effect of paclitaxel and etoposide was cell line- and schedule-dependent. Simultaneous exposure to paclitaxel and etoposide for 24 h produced additive effects in the lung cancer cell line A549 and ovarian cancer PA1 cells, and antagonistic effects in the breast cancer cell line MCF7 and colon cancer WIDr cells. Sequential exposures to paclitaxel followed by etoposide and vice versa produced additive effects in all four cell lines. These results suggest that maximum cytotoxic effects can be obtained with sequential administration, but not simultaneous administration, of paclitaxel and etoposide. These findings may have important clinical implications for this combination.

Schedule-dependent synergism and antagonism between paclitaxel and methotrexate in human carcinoma cell lines.

Paclitaxel and methotrexate are active against a variety of solid tumors. Because of differences in their mechanisms of action and toxicity profiles, the combination of these two agents has clinical potential. Clinical studies of this combination are in progress. We studied the optimal schedule of paclitaxel and methotrexate in combination at various schedules in vitro using human lung cancer A549, breast cancer MCF7, ovarian cancer PA1, and colon cancer WiDr cells. Cells were simultaneously exposed to paclitaxel and methotrexate for 24 h and sequentially exposed to paclitaxel for 24 h followed by methotrexate for 24 h or vice versa. Cell growth inhibition after 5 days was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentration of drug that produced 80% cell growth inhibition (the IC80 level) were analyzed by the isobologram method. The simultaneous exposure to paclitaxel and methotrexate produced additive to antagonistic effects in the A549 and PA1 cells, and antagonistic effects in the MCF7 and WiDr cells. The sequential exposure to paclitaxel followed by methotrexate produced additive effects in all four cell lines. The reverse sequence produced synergistic effects in the A549, MCF7, and WiDr cells, and additive effects in the PA1 cells. These findings suggest that a sequential administration of methotrexate followed by paclitaxel may be the appropriate schedule for this combination. On the basis of the observed in vitro synergism, further in vivo and clinical studies are necessary to clarify the toxicity and proposed antitumor effects of this schedule.

Schedule-dependent interaction between raltitrexed and 5-fluorouracil in human colon cancer cell lines in vitro.

Raltitrexed (Tomudex) is a novel thymidylate synthase inhibitor with significant activity against advanced colorectal cancer. We studied the cytotoxic interactions of raltitrexed and 5-fluorouracil (5-FU) in four human colon cancer cell lines on various schedules. The cell growth inhibition after 5 days was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxic interactions at the IC80 level were evaluated by the isobologram method. Simultaneous exposure to raltitrexed and 5-FU for 5 days produced additive to synergistic effects in Colo201 cells, and produced additive effects in Colo321, LoVo, and WiDr cells. Simultaneous exposure to raltitrexed and 5-FU for 24 h produced additive effects in Colo201, LoVo, and WiDr cells, and produced antagonistic effects in Colo320 cells. Sequential exposure to raltitrexed for 24 h followed by 5-FU for 24 h produced additive effects in Colo201, Colo320, and LoVo cells, and produced antagonistic effects in WiDr cells. The reverse sequence produced additive effects in Colo201 cells, and produced antagonistic effects in Colo320, LoVo, and WiDr cells. Simultaneous exposure to raltitrexed and 5-FU for 4 h and sequential exposure to raltitrexed for 4 h followed by 5-FU for 4 h with a 20-h interval produced additive effects, while the reverse sequence produced antagonistic effects in LoVo and WiDr cells. These findings suggest that the simultaneous administration of raltitrexed and 5-FU or the sequential administration of raltitrexed followed by 5-FU may be the optimal sequence, while the reverse sequence may be inappropriate. Preclinical and clinical studies of the simultaneous administration of raltitrexed and 5-FU and the sequential administration of raltitrexed followed by 5-FU are required to better understand the antitumor, toxic, and pharmacokinetic interactions of this combination in order to develop the combination chemotherapy of raltitrexed and 5-FU.

Expression of inducible nitric oxide synthase in human thyroid papillary carcinomas.

To evaluate the relationship between nitric oxide (NO) and carcinogenesis, we assayed 4 human thyroid papillary carcinomas (TPC) and 3 normal thyroid glands for the presence of inducible nitric oxide synthase (iNOS). Using an antibody against iNOS, we observed immunohistochemical staining if iNOS in the TPC samples, but not in normal thyroid. When we incubated TPC samples with antibodies against both iNOS and human leukocyte antigen (LCA), a macrophage marker, we found that while most TPC cells were stained with anti-iNOS antibody, only a few were stained with both. Reverse transcription polymerase chain reaction (RT-PCR) showed that iNOS mRNA was expressed in the samples of TPC, but not in normal thyroid. The sequence of iNOS message in the TPC samples was identical to that previously detected in a human colon cancer cell line. These results suggest that iNOS in human TPC is mostly derived from tumor cells, rather than macrophages, and that it may play a direct role in carcinogenesis.

Fibrinolysis and fibrinogenolysis in patients with thrombotic disease.

In order to assess the fibrinolytic state in thrombotic disease, plasma levels of fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) were measured in 126 patients with a variety of thrombotic diseases. Mean plasma concentrations of both FbDP and FgDP were significantly elevated in patients with thrombotic disease as compared with healthy subjects. Plasma concentrations of FgDP were positively correlated with FbDP (r = 0.667, P less than 0.001). When analysed according to the disease categories, the magnitude of elevations of FbDP and FgDP was most prominent in venous thrombotic disease such as deep vein thrombosis and pulmonary embolism. These findings indicate that fibrinolysis is accelerated in patients with thrombotic disease and that fibrinolysis is frequently accompanied by some fibrinogenolysis in these patients.

Presence of mRNA for vasoactive intestinal polypeptide (VIP) and its receptor in the rat inner ear.

Although mechanisms regulating inner ear fluid have not been yet elucidated, control of blood flow has been thought to be of great importance. Vasoactive intestinal polypeptide (VIP) was the first neuropeptide demonstrated in cerebrovascular nerves. To study the possible role of VIP in regulation of inner ear fluid, we investigated the presence of mRNA for VIP and VIP receptor in the rat inner ear using a reverse transcription-polymerase chain reaction (RT-PCR) method. A single band of the size expected for VIP and its receptor was detected in mRNA from the rat inner ear by using primers specific for VIP and the receptor. The nucleotide sequences of the subcloned RT-PCR products were identical to those of rat VIP and the rat lung VIP receptor. These results indicate that both VIP and VIP receptor are expressed in the inner ear of the rat and suggest that VIP may be implicated in regulation of fluid in the inner ear.

C-type natriuretic peptide-like immunoreactivity in the rat inner ear.

C-type natriuretic peptide (CNP) is a member of the atrial natriuretic peptide family (ANP family). The family also includes ANP and brain natriuretic peptide (BNP). These peptides regulate the homeostasis of body fluid and blood pressure as a neuropeptide in the central nervous system as well as a cardiac hormone in the periphery. We have recently reported the expression of CNP mRNA in the inner ear. To assess the possible physiological role of CNP in the inner ear, we investigated the localization of CNP peptide in the rat inner ear by immunohistochemistry at the light and electron microscopic level. CNP-like immunoreactivity was widely distributed in the secretory and the neuronal portion of the inner ear, i.e. the spiral ligament, the dark cell region of the utriculus, the epithelium of the endolymphatic sac, the spiral ganglion cells and the vestibular ganglion cells. The results suggest that CNP may play a role in the homeostasis of the perilymph and endolymph and may also influence nerve activities in the inner ear.

Mitral valve prolapse related to geometrical changes of the heart in cases of progressive muscular dystrophy.

The significance of geometrical changes of the heart for the development of mitral valve prolapse (MVP) was studied by echocardiograms and chest x-ray films in 58 cases of progressive muscular dystrophy (PMD). The incidence of MVP was significantly higher (p less than 0.001) in cases where the thoracic spine was straight or lordotic compared with cases of kyphotic thoracic spine. The flattening of the thorax associated with deformation of the thoracic spine was correlated with the left atrial dimension and left ventricular dimension (r = 0.62, r = 0.37, respectively; p less than 0.001), and MVP developed predominantly in cases with flattened thorax and small left atrial or left ventricular dimensions. The left atrial and left ventricular dimensions were significantly smaller in cases with MVP compared to cases without MVP (p less than 0.001, p less than 0.005, respectively). When both the left atrial and the left ventricular dimension shortened to certain levels, MVP was observed in almost all cases. From these results, it was suggested that the portion from the left atrium to the left ventricle was pressed by the forward bending of the thoracic spine, and the subsequent geometrical changes of the mitral ring and the left ventricle could produce redundancy of the chorda tendinea of the mitral valve, resulting in the occurrence of MVP.

Experimental study on Meniere's disease.

Experimental hydrops caused by underabsorption of endolymphatic fluid is a model of remissional stage of Meniere's disease. In this study, another type of model, ie, hydrops caused by overproduction of endolymphatic fluid, was accomplished by applying various pressures into scala media through a micropipette via stria vascularis. This type of hydrops could be a model of attacks of Meniere's disease. By using two types of the model, effects of glycerol administration and of opening the endolymphatic sac were discussed.