Inactivation of Crk SH3 domain-binding guanine nucleotide-releasing factor (C3G) in cervical squamous cell carcinoma.

C3G, a Crk SH3 domain-binding guanine nucleotide-releasing factor functions as a guanine nucleotide exchange factor for Rap1. It is activated via the Crk adaptor protein and plays an important role in transducing signals from receptors on the cell surface to the nucleus via the Ras/Raf/MAPK signal transduction pathway. However, since the experimental data result in pleiotropic effects in the cascade manner, its precise function remains unclear. Here we examined the C3G expression in cervical squamous cell carcinomas and found a marked decrease in the expression of C3G in a high incidence of said samples. In addition, we also demonstrated frequent hypermethylation of C3G, which resulted in an inactivation mechanism of the gene. Clinical and pathologic data failed to show any relationship between the human papillomavirus infection and the down-regulation of C3G. These results indicate that inactivation of C3G by de novo methylation plays an important role in the development of cervical squamous cell carcinoma.

Insulin resistance and impaired myocardial fatty acid metabolism in dialysis patients with normal coronary arteries.

We investigated whether insulin resistance is associated with impaired cardiac fatty acid metabolism in maintenance hemodialysis patients without coronary artery disease. We studied 55 nondiabetic (63+/-11 years old) and 51 diabetic (61+/-10 years old) hemodialysis patients with normal coronary arteries, using single-photon emission computed tomography (SPECT) with an iodinated fatty acid analogue, iodine-123-beta-methyl iodophenyl-pentadecanoic acid ((123)I-BMIPP), to evaluate cardiac fatty acid metabolism. SPECT imaging was graded regionally from 0 (normal) to 4 (absence of tracer) to calculate a summed score for 17 left ventricular segments. Insulin resistance was determined using the homeostasis model assessment index of insulin resistance (HOMA-IR). HOMA-IR correlated with summed BMIPP score in nondiabetic and diabetic patients. Stepwise multiple regression analysis showed that HOMA-IR was independently associated with BMIPP summed score in nondiabetic (beta=0.774, t=9.218, P=0.0001) and diabetic patients (beta=0.792, t=9.079, P=0.0001). Left ventricular ejection fraction was lower in nondiabetic subjects with BMIPP summed score of at least 6 plus HOMA-IR of at least 4 than in others with lower values for both assessments (53.1+/-13.8%, n=20 vs 67.7+/-9.1%, n=23, P=0.0002); this was also true in diabetic subjects (50.9+/-15.2%, n=24 vs 71.0+/-13.6%, n=11, P=0.0007). Association between insulin resistance and impaired cardiac fatty acid metabolism may contribute to left ventricular dysfunction in patients with maintenance hemodialysis without coronary diseases.

Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues.

The plasminogen activation (PA) system may participate in cancer invasion and metastasis. A series of breast cancer tissue specimens was analysed using in situ hybridization and immunohistochemistry. Urokinase-type plasminogen activator (u-PA) mRNA was detected in cancer cells and fibroblasts adjacent to them and its expression was found to be more intense in invasive than in intraductal regions. In invasive but not in intraductal regions, especially those with abundant stroma, plasminogen activator inhibitor-1 (PAI-1) mRNA was observed in cancer cells, fibroblasts, macrophages, and endothelial cells, and PAI-2 mRNA was present in cancer cells, and fibroblasts, macrophages, and lymphocytes around them. These PAI-1- and PAI-2-positive cancer cells were localized at the periphery of the invasive front. Immunohistochemistry yielded basically similar results. A retrospective study of surgically resected breast cancers from 73 patients revealed significant clinical differences associated with u-PA and PAI-2 expression in cancer cells, associated with a poor and a good prognosis, respectively. These findings indicate that breast cancer cells and fibroblasts express u-PA initially and then its inhibitors, and that this process is related to invasion. Expression of u-PA and PAI-2 in cancer cells themselves may serve to up-regulate and limit PA-mediated invasion and metastasis, respectively.

Control of the thermal lensing effect with different pump light distributions.

We built a 32-laser-diode-formed virtual point source pumping system and achieved different pump light distributions from central intense to central uniform and central depressed. Continuous wave TEM(00) operations of a Nd:YAG laser were performed under these pump light distributions and their thermal lensing effects were estimated. Results show that the operation under central depressed pump light distribution has the lowest thermal lensing effect and can provide the highest output power, which agrees with the results derived from the theoretical calculation with the heat conduction equation.

[Dynamic Gd-DTPA-enhanced MR imaging in evaluation of the function of transplanted kidneys].

The purpose of the study was to evaluate the potential of dynamic Gd-DTPA-enhanced MR imaging in assessing of the function of transplanted kidneys. Dynamic MR imaging was performed using the GRASS pulse sequence in 21 subjects (56 examinations), and in each examination 30 images were obtained after a bolus injection of Gd-DTPA. Imaging findings including signal increase and/or signal drop in cortex, signal drop in medulla, and signal drop in calyces were utilized for the evaluation of renal function. Renal function was also evaluated using the following parameters: Ta, time between the initial observation of signal increase in cortex and signal drop in medulla. Tb, time between the initial observation of signal drop in medulla and in calyces; Tc, Ta + Tb, and Max. C/M, maximum ratio of signal intensity between cortex and medulla among 30 images. In the group of patients with normal graft function (Ccr > or = 60ml/min), all imaging findings were observed. In the group of patients with mild graft dysfunction (30ml/min < or = Ccr < 60ml/min), all imaging findings but signal drop in cortex were observed. The time parameters of Ta, Tb, and Tc were significantly larger, and Max. C/M was significantly smaller than those observed in the group with normal graft function. In the group of patients with severe graft dysfunction (Ccr < 30ml/min), almost none of the findings were observed. The investigation in patients in the postoperative state revealed a close correlation of the imaging findings and the values of parameters with changes in graft function over time. The results indicated the usefulness of the method in semiquantitative evaluation of graft function, including that of patients in postoperative state. This study suggests that dynamic Gd-DTPA-enhanced MR imaging could be a valuable method for the management of transplanted kidneys.

[A clinical study of recurrent breast cancer].

In a recent 14-year period from December 1978 to December 1993, 180 patients with breast cancer were treated at the hospital, and out of these 180 patients, 38 patients (21.1%) who had a recurrent breast cancer were clinically evaluated. The first recurrence occurred in the local skin in 13 patients, regional lymph node in 1, bone in 13, lung in 4, liver in 4 and in other areas in 3. Histologically, the incidence of scirrhous carcinoma recurrence was higher than that of papillotubular carcinomas. There was no recurrence in mucinous carcinomas. The frequency of recurrence became higher in accordance with TNM classification, namely, 6.7% in Stage I, 23.7% in Stage II, 37.5% in Stage IIIa, and 47.1% in Stage IIIb cancers. An increasing tendency in the recurrence rate was also noted with an increase of tumor diameter or lymph node metastasis. Otherwise, there was no difference in cumulative survival after recurrence between TNM classification. There were no correlations between the estrogen receptor and incidence of recurrence or 5-year survival rate. The disease free interval (DFI) was less than 2 years in about 60% of the recurrent cases. DFI of bone metastasis was longer than for the other sites of recurrence. The survival rate increased according to prolongation of DFI. After through evaluation of these results, one should pay close attention to follow-up after mastectomy.

[A new cellulose-barium gel for computed tomography of the esophagus].

A new oral contrast agent for CT of the esophagus was evaluated for its ability to coat the esophageal lumen in 12 patients with esophageal diseases. This agent is made of carboxymethyl cellulose sodium, potato starch and low-density barium, and has remarkably high viscosity of 13600 centipoise. The esophagus was divided into proximal, mid, and distal segments to analyze the effectiveness of lumenal opacification. The average percent opacification of these segments was 78.7, 86.4, and 63.4%, respectively, for all patients. This agent is expected to play a possible role in the CT diagnosis of esophageal cancer by helping to determine the indications for radical surgery and/or radiotherapy.

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.

[Cyclosporin levels in semen of renal transplant patients].

Cyclosporin (CsA) has powerful immunosuppressive properties, and its recent application to organ transplantation has resulted in markedly improved graft survival. However, CsA has certain adverse side effects, the most notable being nephrotoxicity and hepatotoxicity. Among other untoward effects, little is known about the effects of CsA on the reproductive organs. Since good graft survival is now expected with CsA, the long term effect of the drug on the gonads should be monitored carefully, particularly for young recipients. Prior to investigation of the effect of CsA on the testis, the level of CsA in semen of the adult renal transplant patients were measured. Twelve samples of semen from eight recipients, mean age 35 (SD 9), were collected by masturbation in the morning just before taking CsA after more than 5 day abstinence, and it was frozen at -80 degrees C. They had been taking immunosuppressants that were a combination of either CsA and prednisolone, or CsA, azathioprine and prednisolone for 5 to 63 months. The dose of CsA was 3.7 mg/kg to 5.0 mg/kg. The range of the level of serum creatinine was 1.1 mg/dl to 2.9 mg/dl. CsA in whole blood was measured by high performance liquid chromatography (HPLC), and CsA in semen was also measured by HPLC after extracting CsA with diethylether. The level of CsA in semen was 27 ng/ml to 165 ng/ml and that in whole blood was 51 ng/ml to 133 ng/ml. The relation between both levels was linear (r = 0.68, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[A study on the hemodialysis period following cadaveric kidney transplantation--a comparison between two centers sharing cadaver donor kidney pairs].

To study the hemodialysis period following cadaveric renal transplantation, we reviewed 18 consecutive cadaver donor kidney pairs of which one kidney was transplanted at Shiga University of Medical Science (SUMS) while the contralateral kidney was transplanted at Kyoto Prefectural Medical University (KPMU) between January 1983 and September 1988. Of these 18 pairs, graft function was satisfactory and the recipient was free from hemodialysis in 13 pairs. However, the postoperative hemodialysis periods were different between the two centers. To clarify the factors causing this difference, recipient factors, such as age, HLA matching, total ischemic time of the graft (TIT), immunosuppression, onset of diuresis and postoperative complications were studied. There were no significant differences in age, HLA matching, or immunosuppression. TIT of cases at SUMS was significantly longer than that of cases at KPMU. The postoperative hemodialysis period at SUMS was 25.8 days and that at KPMU was 17.8 days, however, the difference was not statistically significant. The results of this study indicate that TIT seems to be a major factor relating to delayed graft function in cadaveric renal transplantation.

Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.

Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequence deduced from the isolated cDNAs revealed several interesting features, including hydrophobic signal and transmembrane domains that flank an extracellular region containing six potential attachment sites for glycosaminoglycan side chains. The structure also contains a short hydrophilic cytoplasmic tail sequence homologous to previously reported actin binding domains. Binding of basic FGF to cells expressing the binding protein could be inhibited by heparin and heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. In addition to binding basic FGF, this protein or related surface proteins may function as an initial cellular attachment site for other growth factors and for viruses, such as herpes simplex virus.

[Pneumocystis carinii pneumonia in renal transplant recipients treated with cyclosporin].

Four cases of pneumocystis carinii pneumonia occurred among 38 renal transplant recipients. Diagnosis was confirmed by cyst concentration technique in 2 cases. The other 2 cases were clinically, from the rapid improvement of fever, pulmonary infiltrates and hypoxia following a therapeutic trial of high dose sulfamethoxazole-trimethoprim. All patients responded to treatment with high dose sulfamethoxazole-trimethoprim. Three patients survived pneumocystis carinii pneumonia, but i died due to aspergillosis. One patients showed serological evidence of concomitant cytomegalovirus infection.

[Effectiveness of FUT-175, protease inhibitor, as an anticoagulant to hemodialysis].

(6-Amidino-2-naphthyl 4-guanidino benzoate) dimethanesulfonate (FUT-175), a protease inhibitor, has been reported to be an effective anticoagulant during hemodialysis without heparin. The anticoagulant activity of FUT-175 is also reported to be short. We applied FUT-175 to 33 patients who were undergoing hemodialysis and susceptible to bleeding, to avoid the use of heparin. The concentration and anticoagulant activity of FUT-175 were relatively stable during hemodialysis. A 20-40 mg/h dose of FUT-175 prolonged coagulation time sufficiently in the instrumental blood of the extracorporeal circuit but not in the systemic blood. Its anticoagulant activity decreased immediately after hemodialysis. Therefore, we could manage all patients without any bleeding trouble during hemodialysis with FUT-175 as an anticoagulant. Although there were side effects of FUT-175, such as nausea, vomiting, itching and eruption, they were not serious, and FUT-175 could be administered without interruption. FUT-175 seems to be useful as an anticoagulant during hemodialysis for patients susceptible to bleeding.

[A case of left inferior vena cava discovered by chance at nephrectomy for renal transplantation].

A case of left inferior vena cava, in a 35-year-old woman, discovered by chance at nephrectomy for renal transplantation is reported. She was admitted to our hospital as a donor of the kidney to her son. She had no remarkable physical signs on chest or abdomen. All laboratory tests were within normal limits. Intravenous pyelogram showed no major abnormality except for the lower position of left kidney than right one. Abdominal aortogram and selective renal arteriogram revealed no abnormality. Bilateral renal arteries were single. Left nephrectomy for transplantation was performed on April 13, 1983. In this operation the left sided inferior vena cava was discovered by chance. Inferior vena cavography was taken postoperatively. It showed the left sided inferior vena cava, type C. Knowledge of the abnormalities of the left renal vein and inferior vena cava is of surgical importance for the urologist when renal transplantation is being considered.

Immunohistochemical study of the cells infiltrating human renal allografts by the ABC and the IGSS method using monoclonal antibodies.

Sixty renal allograft tissues obtained from 29 patients were stained with hematoxylin and eosin. These tissues were histologically classified into 4 patterns according to the distribution pattern of the infiltrating cells: normal, focal, focal-diffuse, and diffuse types. Clinical signs of acute rejection were observed in 88% of the patients with the diffuse type infiltration, and 83% of those with the focal-diffuse type infiltration but in only 13% of those with the focal type infiltration. Twenty-four renal allografts were analyzed by the ABC and the IGSS methods using monoclonal antibodies. The number of T cells (Leu 1) accounted for about 80% of the total number of infiltrating cells; 2-8% of the cells were B cells (Leu 12); about 10% were NK/K cells (Leu 7); and 4-6% were monocytes/macrophages (Leu M3). As to helper/inducer T cell (Leu3a) and killer/suppressor T cell (Leu2a), which are T lymphocyte subsets, there were more Leu3a- than Leu2a-positive cells in focal type tissue, but there were more Leu2a- than Leu3a-positive cells in focal-diffuse and diffuse type tissue. In most cases that developed clinical signs of acute rejection, there were more Leu2a- than Leu3a-positive cells. The Leu3a/Leu2a ratio in most of the AZA-administered cases dropped immediately after the transplantation and maintained a low value, but in the CSA-administered cases it decreased gradually post-transplant.

Warfarin-griseofulvin interaction.

A 61-year-old man with a mitral valve replacement who had been therapeutically anticoagulated with warfarin for the previous 12 months had a decrease in his prothrombin time after starting treatment with griseofulvin. Over a three-month period his warfarin dose was gradually increased. An overall increase of 41 percent was needed to maintain therapeutic anticoagulation. A reduction in the griseofulvin dose resulted in an increase in the prothrombin time and, subsequently, a lowering of the warfarin dose. No other potential causes for the decreased response to warfarin were identified. While there previously has only been limited documentation of this interaction, it appears that a substantial decrease in the response to warfarin can occur following initiation of griseofulvin therapy. Immediate adjustment of the warfarin dose at the onset of griseofulvin therapy is not needed. However, the prothrombin time should be monitored at weekly intervals and the dose of warfarin increased according to the patient's response.

Simultaneous detection of B-cells and T-cells by a double immunohistochemical technique using immunogold-silver staining and the avidin-biotin-peroxidase complex method.

A new double immunohistochemical technique for the simultaneous detection of B-cells and T-cells was investigated, using tissue preparations obtained from human axillary lymph nodes and rejected renal allografts. The specimens were immunostained first for the demonstration of B-cells, by the immunogold-silver staining (IGSS) method using Leu-12 monoclonal antibody, and then for T-cells by the avidin-biotin-peroxidase complex (ABC) method using Leu-1 monoclonal antibody. With the present methods, both B-cells and T-cells were clearly detected and distinctively identified without cross-linking of antibodies or double reaction of enzymes.