Analysis of fine-needle aspirates for HPV by PCR may be useful in diagnosis of metastatic gynecologic malignancies. Human papillomavirus (HPV) DNA has been shown by molecular hybridization studies to persist in both recurrent and metastatic disease in tumors of the female genital tract. We report here the use of the polymerase chain reaction to identify HPV DNA in material from fine-needle aspirates (FNA) of recurrent or metastatic lesions to document the primary malignancy arising in the lower genital tract. Fine-needle aspirates of suspected recurrent or metastatic tumors were obtained from nine patients with carcinoma of the lower genital tract and five patients with malignancies that have not been associated with HPV. DNA was extracted from the FNA and tissue block, when available, and amplified with HPV 6, HPV 16, and HPV 18 specific primers. In eight of the nine tumors from the lower genital tract, HPV DNA was identified in both the primary and metastatic lesions. In every case the HPV genotype was identical. One cervical carcinoma and five non-HPV associated tumors were negative for papillomavirus DNA. This study demonstrates that molecular hybridization techniques can be useful in identifying the source of a metastasis and have the potential to diagnose the presence of metastatic disease by detecting HPV DNA even when the cytologic criteria are equivocal.


Phyllodes tumor in a patient with a preexistent fibrocystic disease. A phyllodes tumor appeared at the site of a resected fibrocystic disease focus. Administration of danazol resulted in temporary regression, but the tumor resumed growth. Relative hyperestrogenism also continued to increase in spite of continued long-term administration of danazol. After 12 months a simple mastectomy was performed. Most of the resected mass consisted of the phyllodes tumor, but it also contained fibrocystic disease.


Effects of pertussis toxin treatment on human natural killer cell function. Membranes from highly purified natural killer (NK) cells were ADP ribosylated by treatment with pertussis toxin (PTX). PTX treatment resulted in a single band of 32P incorporation at M(r) 41,600. PTX treatment of NK cells diminished their ability to lyse K562 tumour cells by about 50%. However PTX treatment had no measurable effect on cAMP levels in NK cells. PTX pretreatment also had no effect on the ability of target cells to induce phosphoinositide turnover or on the ability of the NK cells to conjugate with the K562 tumour cells. Movement toward the chemoattractants interleukin-2 (IL-2) and formylmethionylleucylphenylalanine (FMLP) was significantly inhibited indicating that a PTX substrate in NK cells may be involved in the transduction of signals which are involved in cell motility.


Expression of nerve growth factor receptor immunoreactivity on follicular dendritic cells from human mucosa associated lymphoid tissues. Nerve growth factor (NGF) was originally considered as a trophic factor for peripheral sympathetic and sensory neurones; however, recent reports indicate that NGF may induce proliferation of immune and haematopoietic cells. Histochemical studies conducted in human spleen and lymph nodes have suggested the presence of NGF receptor (NGF-R) immunoreactive elements in secondary follicles; however the nature of the cells bearing the NGF-R in lymphoid tissue has not been determined. In this paper we report the results of an immunohistochemical study conducted on mucosa associated lymphoid tissue. Using a specific monoclonal antibody to human NGF-R (mAb 20.4) we observed an NGF-R-immunoreactive population in all secondary lymphoid follicles examined. Double immunostaining revealed that this population was composed of follicular dendritic cells (FDC); lymphoid cells within the germinal centres did not appear to be 20.4 immunoreactive. Cell suspensions from tonsillar follicles also contained NGF-R immunopositive dendritic cells which were enriched by a 20.4 labelled magnetic bead procedure, revealing cells with the morphological characteristics of FDC. Mononuclear cells from human peripheral blood did not contain any NGF-R-immunoreactive elements using our techniques.


Alteration in human mononuclear leucocytes following space flight. Reduced in vitro mitogen-stimulated proliferative responses have routinely been observed from astronauts' mononuclear leucocytes following space flight. This study investigated the effect of space flight on subpopulations of peripheral blood mononuclear cells from 30 shuttle astronauts prior to launch, upon landing and 3 days after flight. The total number of peripheral blood leucocytes, granulocytes and monocytes were increased after space flight (5.7 +/- 0.2 versus 7.0 +/- 0.2; 3.1 +/- 0.1 versus 5.0 +/- 0.1; and 0.16 +/- 0.02 versus 0.25 +/- 0.28 x 10(3) cells/mm3, respectively) whereas lymphocytes were decreased (2.2 +/- 0.1 versus 1.7 +/- 0.1 x 10(3) cells/mm3). Flow cytometry analysis on Ficoll-Hypaque isolated mononuclear cells upon landing revealed significant decreases in T-inducer (CD4+, Leu-8+; 32 +/- 2 versus 23 +/- 2%) and T-cytotoxic lymphocytes (CD8+, CD11b-; 17 +/- 1 versus 12 +/- 1%), and increases in monocytes (CD14+; 13 +/- 1 versus 21 +/- 1%) compared to pre-flight and post-flight samples whereas B cells (CD19+), T-helper (CD4+, Leu-8-) and T-suppressor (CD8+, CD11b+) populations did not change. Additional phenotypic analysis of these mononuclear leucocytes from 10 crew members upon landing revealed a reduction in natural killer (NK) cells (CD16+ or CD56+; 9 +/- 1 versus 3 +/- 1%) and an increase in monocytes that were negative for insulin and insulin-like growth factor-1 (IGF-1) receptor expression. Flow cytometric analysis indicated these hormone receptor negative monocytes were smaller and less granular than receptor positive monocytes. Therefore, a novel population of monocytes may be released into the peripheral blood during the stress of space flight or upon landing. These findings may explain some of the diverse in vitro immunological and endocrine changes observed in crew members following space flight.


Haplotypic origin of beta-chain genes expressed by human T-cell clones. A contribution of allelic variation of T-cell receptor (Tcr) genes to the immune response has not been studied. Here we report that the presence of insertion-deletion-related polymorphisms (IDRP) of the Tcr beta chain (Tcrb) can be utilized to distinguish the parental origin of the gene complex that undergoes rearrangement in individual T-cell clones. Phytohemagglutinin stimulated clones from an individual heterozygous for an IDRP located between the variable (V) and diversity (D)-joining (J) region genes were studied for the presence of V to DJ rearrangements in each of the two parental chromosomes. Results indicate that single rearrangements were present in the majority of clones, in contrast to the double rearrangements of D to J genes that were generally present. In this individual, V to DJ rearrangement also occurred with different frequencies on each of the two germline genes. IDRP clonotyping of the Tcrb complex should prove generally applicable to the study of the influence of allelic variation of Tcrb genes in selection of the expressed T-cell repertoire.


Priming effects of leukotriene B4 on endothelial cell injury induced by TPA-activated leukocytes. Among arachidonic acid metabolites, leukotriene B4 (LTB4) plays an important role in inflammation, such as in the activation, adhesion, chemotaxis, and invasion of leukocytes. In this paper, we examined the effect of LTB4 on endothelial cell injury induced by polymorphonuclear leukocytes (PMNLs). 51Cr release, a marker of cellular injury, was elicited from prelabeled endothelial cells when the cells were cocultured with PMNLs activated by phorbol ester (TPA, 12-O-tetradecanoyl-phorbol-13-acetate). Under this condition, pretreatment of PMNLs with LTB4 enhanced their injury in a dose-dependent manner (0.2-2 microM). However, LTB4 alone at any dose could not induce any cellular injury. We also determined the amount of active oxygen species produced by PMNLs in response to TPA. The intensity of luminol-dependent chemiluminescence, a marker of active oxygen production, in PMNLs was also increased by pretreatment with 1 microM LTB4. These data suggest that LTB4 enhances endothelial cell injury by the priming effect on active oxygen production in activated PMNLs.


Prevention and reversal of experimental colitis by a monoclonal antibody which inhibits leukocyte adherence. The role of neutrophils in the pathogenesis of acute colitis was investigated using a rabbit model. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid in 30% ethanol. Myeloperoxidase activity was measured at various times after induction of colitis as an index of neutrophil infiltration, and this was confirmed by histology. The permeability of the colonic epithelium to [51Cr]EDTA was also measured at various times after induction of colitis. The most marked increase in neutrophil infiltration of the colon occurred during the period 3-6 h after induction of colitis. This was also the period in which the greatest increase in colonic permeability was observed. Pretreatment with a monoclonal antibody (IB-4) directed against the leukocyte adhesion molecule, CD18, markedly suppressed neutrophil infiltration into the colonic tissue after induction of colitis. This pretreatment also significantly reduced the extent of epithelial injury. Administration of IB-4 to rabbits 12 h after induction of colitis resulted in a rapid decline in tissue myeloperoxidase activity. When measured 12 h after IB-4 administration (3 mg/kg), colonic myeloperoxidase activity was reduced by about 80% compared to the control group treated with the vehicle. These results are consistent with the hypothesis that neutrophils contribute significantly to the epithelial dysfunction that characterizes colitis and suggest that antibodies directed against adhesion molecules may represent a novel approach to the treatment of intestinal inflammatory disorders.


Resting metabolic rate and body composition of Pima Indian and Caucasian children. Since a low metabolic rate (for a given body size and body composition) is a risk factor for body weight gain and obesity is prevalent among Pima Indians, we have tested whether Pima Indian children have a low resting metabolic rate (RMR) as compared to Caucasian children. Body composition (bioelectrical resistance) and RMR were measured in 43 Pima Indian children (22 male/21 female, mean +/- s.d. 9.9 +/- 1.1 years) and 42 Caucasian children (21 male/21 female, 9.7 +/- 1.2 years). Pima children were taller (143 +/- 9 vs. 137 +/- 8 cm, P less than 0.001), heavier (48.6 +/- 15.8 vs. 32.9 +/- 7.8 kg, P less than 0.001) and fatter (39 +/- 10 vs. 24 +/- 7% fat, P less than 0.001) than Caucasians. Absolute values of RMR were higher in Pimas than in Caucasians (6234 +/- 1201 vs. 5171 +/- 715 kJ/day, P less than 0.001), but similar when adjusted for differences in body size, body composition and sex. Contrary to our hypothesis, we did not find a decreased RMR in Pima children when compared to Caucasian children. Therefore, the high prevalence of obesity in Pima children at age 10 suggests that excess energy intake and/or low levels of physical activity might be the major aetiological factor. However, this study does not exclude the possibility that a low metabolic rate might be a predisposing factor at an earlier age.


Estimation of computerized tomography derived abdominal fat distribution. Measurements of intra-abdominal fat (IAF) may be important since it is associated with numerous metabolic disorders. The relationship between computerized tomography (CT) measured fat distribution and densitometry measures was investigated in a sample of 61 male Caucasian subjects, aged 18 to 30 years with varying adiposity. Regression models were developed for estimating CT-derived fat of 40 men to estimate IAF. Two equations were developed to estimate IAF. The first used only anthropometric measures. Waist circumference and log chest ratio entered the equation and accounted for 67% of the variance. The second model included densitometry-measured percentage fat with the centred product of waist and hip circumferences, accounting for 73% of the variance. Regression equations were also developed to estimate subcutaneous fat area so that the ratio of IAF to subcutaneous fat might be estimated. Although subcutaneous fat could be estimated, the ratio between IAF and subcutaneous fat could not be estimated accurately. A validation of all regression equations developed for male subjects who also completed using a separate validation sample (n = 21). Only the studies with sample characteristics similar to those found in the validation sample validated satisfactorily. Results indicate that anthropometric and densitometry measures cannot be used to estimate CT-derived abdominal fat with precision, however they may be of value in health risk screening of individuals with high levels of IAF. Proper selection procedures with regard to age, adiposity, and morbidity must be used.


Nitrogen metabolism in obesity induced by monosodium-L-glutamate in rats. We have studied the effects of subcutaneous administration of monosodium-L-glutamate (MSG) to neonatal rats on nitrogen metabolism and on general parameters at several intervals after MSG treatment. As MSG-treated rats were hypophagic, all experiments were performed both in control rats pair-fed with the MSG-treated rats and in control rats fed ad libitum. Lee index, total serum lipids and weight of the epididymal fat depots were higher in MSG-treated rats. Body and tissue weights and the amount of protein in several tissues were lower in adult MSG-obese rats than in control rats. Locomotor activity was decreased following MSG administration. Creatinine clearance was diminished by about 50% in rats treated with MSG. Urinary nitrogen and urea excretion were lower, except at four weeks, and serum urea was higher in MSG-obese rats. Considering liver size, urea synthesis by isolated hepatocytes and urea cycle enzyme activities were increased in weanling MSG obese rats and diminished in adult MSG-obese rats when compared with ad libitum controls but were not changed compared with their pair-fed controls. It is concluded that administration of monosodium-L-glutamate shortly after birth induced an increase in urea synthesis in weanling rats that was followed by a reduction in the amount of tissue proteins, suggesting that more amino acids were used for lipid synthesis and urea production in treated rats. The accelerated amino acid degradation slowed down in adult MSG-obese rats which showed an in vitro capacity to synthesise urea similar to that of their pair-fed controls.


Prevalence of obesity: a comparative survey in France, the United Kingdom and the United States. We investigated the differences in the prevalence of obesity between France, the United Kingdom and the United States in 1988. The analysis was made on a total sample of 5580 subjects, representative of the population aged 16-50 years in the three countries. The same questionnaire was used in all three countries. Body mass index (kg/m2) was used to assess corpulence. Significant differences in the prevalence of obesity were observed among the three countries: 7% of the population in France was obese, 9% in the UK and 15% in the USA (P less than 0.001). There was a strikingly high percentage of very obese women (more than 50% overweight) in the USA (8% of the population) as compared to the two European countries (2% in France and 3% in the UK). In all three countries, obesity was related to sex, age, level of education, marital status, physical exercise and smoking. An inverse association was found between obesity and alcohol consumption in the USA, but not in France nor in the UK. In men, prevalence of obesity remained significantly higher in the USA than in France or in the UK when adjusting for the obesity-related factors. In women, differences in prevalence of obesity between the three countries varied according to the level of exercise, income and alcohol consumption.


Lipoprotein sub-fraction levels and composition in obese subjects before and after gastroplasty. Obesity is frequently associated with several alterations of plasma lipid levels and lipoprotein metabolism. To evaluate the effects of severe obesity and weight loss on plasma lipoprotein sub-class levels and composition 11 grossly obese patients were examined before and six and 12 months after gastroplasty. Plasma lipoproteins were isolated by ultracentrifugation in a zonal rotor under rate flotation conditions. Mean body weight was 121.9 kg before gastroplasty, 97.6 kg after six months, and 90.7 kg after 12 months. Total plasma cholesterol was not affected by the weight reduction. Obese patients were characterized by increased levels of IDL and small dense LDL (LDL3). LDL levels, but not LDL3, were reduced following weight reduction. Plasma apo B levels of obese patients were always higher than in controls and were not affected by the weight reduction. After 12 month's weight loss total HDL cholesterol increased without modification of HDL sub-class cholesterol distribution, which was similar to that of normal controls. Plasma apo AI in obese patients was not affected by changes in body weight, and remained below normal. The percentage protein-lipid composition of LDL2 and HDL3 in obese patients was characterized by a decreased cholesterol ester content before gastroplasty which was normalized 12 months after gastroplasty. The presence of IDL and LDL3 in increased concentrations in severely obese patients may represent a vascular risk factor since similar abnormalities were recently observed in non-obese patients affected with vascular diseases.


Estimation of peak oxygen consumption from a sub-maximal half mile walk in obese females. This study compared peak oxygen consumption estimated from a half mile sub-maximal walk test with oxygen consumption determined from a maximal treadmill test with 96 obese females. A half mile walk was completed as quickly as possible without running. Retests on 71 subjects were completed to determine the reliability of the half mile walk. Hearts rates were obtained for 15 s immediately upon completion of the walks. Stepwise regression to estimate peak VO2 was used with the following variables: walk time (TM), body weight, height, BMI, age, and heart rate (HR). The best equation was peak VO2 (ml/kg/min) = 53.23 - (1.98 x TM) - (0.32 x BMI) - (0.08 x age), r = 0.76, s.e.e. = 2.89 ml/kg/min. The mean difference between determined and estimated peak VO2 in a cross-validation group of subjects who were not included in the validation group was 0.15 ml/kg/min (n.s.), r = 0.77, s.e.e. = 3.01 ml/kg/min, E = 3.09. Reliability for time to complete the half mile walk was r = 0.87 with 0.1 min difference (n.s.). This study indicated the half mile walk to provide an accurate, reliable estimate of peak VO2 which should be useful in the clinical setting.


Activity decreases as percentage overweight increases. The purpose of the present research was to clarify the empirical relationship between percentage overweight and degree of ambulatory activity. Two week activity measurements were obtained, over two studies, using pedometers, from 127 women aged 19 to 55 years ranging from 14% underweight to 99% overweight. Regression analysis indicated that activity decreased by 7.6457 x 10(-4) mph for every 1% increase in percentage overweight from 0.25279 mph associated with zero percentage overweight. These data are in good agreement with data published by Chirico and Stunkard in 1960.16 Implications for small activity increases on obesity and general health are discussed.


Long-term follow-up of patients attending a combination very-low calorie diet and behaviour therapy weight loss programme. We examined the effects of treatment with a very-low calorie diet (VLCD) combined with behaviour modification on weight loss and long-term maintenance of weight loss in 118 of 199 patients who completed eight weeks of VLCD. Those who began therapy in 1984 were surveyed by telephone an average of 3.3 years after ending the VLCD. Questionnaire data included reported weight, exercise, eating, work and sleep habits, emotional factors, and current use of behavioural techniques taught in the programme. Results showed that mean maximum weight loss during the time they attended the programme (average 51.6 weeks) was 31.3 kg, corresponding to a decrease in body mass index of 10.7 kg/m2. At follow-up a regain of 60.9% was reported yielding a net overall weight loss of 13.6 kg and decrease in body mass index of 4.4 kg/m2. Seventy-five per cent of subjects showed only a 37.5% regain of the weight they had lost. Those whose weight loss was better maintained at the time of follow-up reported exercising more, eating fewer high fat foods, and using more of the behavioural techniques taught in the programme. This study provides support for the conclusion that some patients treated with VLCD and behaviour modification can maintain significant weight losses over a relatively long period of time and that specific behaviours relate to this success.


Mechanism of action of cytotoxic antibodies to adipocytes on adipose tissue, liver and food intake in the rat. The aim of this study was to characterize in greater detail the effects of anti-(rat adipocyte plasma membrane) antisera (A/S 83 and A/S 164) that had previously been shown to cause large reductions in total body fat. Both antisera produced lymphocytic infiltration of adipose tissue and reduction of adipocyte numbers seven days after treatment as well as a reduction in food intake and body weight on the first day of treatment. In addition A/S 164 produced a sedative effect for 2-4 h after treatment and induced gross abnormalities of the liver after seven days. Antibody-mediated complement activation was shown to be a critical requirement for all of these effects since animals treated with cobra venom factor (CVF) to deplete serum complement levels showed none of the described effects. Further evidence in support of a role for complement was the large decrease in serum complement levels 12 h after antiserum treatment. The effects of A/S 164 on liver morphology could be successfully dissociated from those on adipose tissue by adsorption of the antiserum with liver membranes. The adsorbed antiserum, which retained in vitro reactivity with adipose tissue but not with liver, induced a significant reduction in adipose tissue mass in vivo whilst the effect on liver morphology was almost completely abolished. Serum free fatty acid and triglyceride levels increased 6-24 h after treatment but then returned to normal, suggesting a very transient release of adipose tissue triglycerides. These results indicate that complement activation is a common pathway for all of the effects produced.(ABSTRACT TRUNCATED AT 250 WORDS)


Transcriptional activity of HIV-1 and HHV-6 in retinal lesions from AIDS patients. This study determined the frequency of multiple viral (HIV-1, HHV-6, and CMV) infections in 26 retinas from 16 AIDS patients. Of the 12 retinas of 26 that tested positive for HIV-1 DNA sequences, seven also were positive for HHV-6 DNA sequences. Four of these seven retinas were culture positive for HIV-1 and two of the four contained CMV DNA sequences and antigens. Using RNA probes, HIV-1 and HHV-6 transcriptional activity was demonstrated in two of the four HIV-1 culture positive retinas. These retinas also contained CMV DNA sequences and antigens. The results demonstrate that more than 35% of AIDS patients suffer from at least two simultaneous viral infections and 15% suffer from three viral infections. The presence of transcriptional activity of HIV-1 and HHV-6 suggests an active infection.


Oxiradical-dependent photoemission induced by a phacoemulsification probe. Oxygen free radical formation by conventional phacoemulsification devices has been postulated as a possible mechanism of corneal endothelial damage during surgery. To test this hypothesis, phacoemulsification probe-induced free radical production was visualized using a single photon-counting camera and an O(2-)-sensitive luciferin derivative, 2-methyl-6-[p-methoxyphenyl-3,7-dihydroimidazo [1,2-a]pyrazin-3-one (MCLA), which allows the visualization of spatial and temporal alterations in free radical production. Within 1 min after starting ultrasound emission, MCLA-dependent chemiluminescence was increased significantly, the intensity of which was maximal at the tip of the probe and tapered along a gradient toward distal portions. The chemiluminescence was suppressed significantly by adding either superoxide dismutase (300 U/ml) or sodium azide (20 mmol/l). By adding deuterium to the medium, MCLA-dependent chemiluminescence significantly increased, suggesting the involvement of singlet oxygen in the reaction.


Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands. The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.


Charge and pH effect on the early events of Epstein-Barr virus fusion with lymphoblastoid cells (Raji). Fusion of Epstein-Barr virus (EBV) with Raji cells was measured after exposure of the virus to neutral or low pH, enzymatic modification of the viral spike glycoproteins, or chemical modification of the target membrane. The relief of octadecylrhodamine (R18) fluorescence self-quenching was used to monitor fusion. Fusion of EBV with Raji cells at pH 5.9 was significantly enhanced compared to that at neutral pH. Treatment of Raji cells with agents known to modify the surface net charge (trinitrobenzene sulfonic acid) totally prevented fusion at a neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Our results demonstrate that EBV is rapidly internalized and then fuses with lymphoblastoid cells in the endocytic vesicles.


Differences in retention and expression of transfected human cytomegalovirus Towne XbaI-E transforming fragment in human cervical and NIH 3T3 lines. Human cervical and NIH 3T3 cells were transfected with the XbaI-E-transforming fragment of human cytomegalovirus strain Towne. Southern blot hybridization showed that 3 of 4 transformed NIH 3T3 cell lines retained only the mtrII subfragment of Towne XbaI-E, but not the mtrIII subfragment. Even though mtrII was retained, no viral transcripts were detected. Analysis of genomic DNAs isolated from three independently derived lines of Towne XbaI-E-transfected human exocervical epithelial cells previously immortalized by human papillomavirus type 16 (CX16-2/Towne-E) revealed the retention of both mtrII and mtrIII subfragments of Towne XbaI-E even after greater than 30 subpassages. Southern blot hybridizations indicated the integration and rearrangement of mtrII as well as mtrIII. Poly (A)+RNA analysis of CX16-2/Towne-E line revealed a 1.9-kb transcript which hybridized to mtr III. In contrast, no viral transcript from the mtrII region was detected in these cells. The pattern of HPV-16 DNA sequences and the profile of RNA transcripts were similar in the parental human exocervical cells (CX16-2) and in the CX16-2/Towne-E cells. Thus far, the CX16-2/Towne-E lines are nontumorigenic in nude mice. This study highlights not only differences in the ability of Towne XbaI-E to transform rodent cells and not human cells but also differences in the retention and expression of mtrII and mtrIII in these cells.


Herpes simplex types 1 and 2: latency in the genital tract of guinea pigs. Guinea pigs were infected with herpes simplex virus (HSV) intravaginally and then sacrificed during latent infection. Virus was recovered from the ganglia, spinal cord and genital tissues by co-cultivation after 1-6 weeks in culture. The virus could not be recovered from the genital tract during the first week of co-cultivation, nor from homogenized genital tissue. Cultivation of genital tissues with acyclovir did not reduce the recovery of HSV. Thus, HSV appeared to establish a truly latent infection in the genital tract and not a persistent infection as previously described.


Effect of protein kinase C inhibitors with different action mechanisms on Epstein-Barr virus replication. 12-0-Tetradecanoyl phorbol-13-acetate (TPA) has been widely known as an activator of Epstein-Barr Virus (EBV) replication and is a direct activator of protein kinase C (PKC). These facts suggest that EBV DNA synthesis might at least partly be dependent upon PKC activity. In this report, the effects of two different types of PKC inhibitors on EBV DNA synthesis were investigated by slot blot hybridization using a biotin-labeled probe. Staurosporine and H-7, inhibitors acting on the catalytic domain of PKC, prevented the growth reduction of P3HR-1 cells harboring EBV genomes and the induction of viral DNA synthesis by TPA. Calphostin C and sphingosine, which have been reported to suppress the enzyme activity by acting on the regulatory domain of PKC, did not exert efficiently effects on cellular growth and viral replication at increasing concentrations of TPA. From these results it is suggested that PKC is involved in the control of viral DNA synthesis in P3HR-1 cells and that for the inhibition of virus growth, it is more effective to suppress the activity of the catalytic domain of this enzyme than acting on the regulatory domain and competing with PKC activators such as TPA for binding.


[Differential diagnosis of breast tumors with eczematous breast changes: two rare cases]. It is reported upon two patients who were presented to us to evaluate suspicious tumors of the breast: A 64-year-old patient had very painful tumors in both breasts measuring four centimeters in diameter with additional eczematous lesion of the left mamilla, which has developed within the last weeks. For many years she suffered of a pseudoxanthoma elasticum (PXE) of the skin combined with arterial occlusion disease. Histologically these tumors showed necrosis and typical signs of PXE, which is to our knowledge the first case reported in literature. A 79-year-old patient showed an inverted eczematous nipple, combined with a retromamillar painless tumor of the left breast. The histology was Paget's disease with an underlaying ductal carcinoma. --The clinical picture and the diagnosis are presented, followed by the discussion and the differential diagnosis.


[Ulcerated breast cancer of the young patient: a case report]. A 29-year-old woman presented with an exulcerating cancer of the right breast. She underwent a total mastectomy and axillary clearance which revealed a pathological stage of T4 N+ (21/23) M0. She received chemotherapy followed by radiation therapy and by chemotherapy again. 30 months later, a contralateral breast cancer was diagnosed. A close team-work of gynaecologist, plastic surgeon, medical and radiation oncologists allowed to combine the operation with the reconstruction of both breasts together with chemotherapy and irradiation of the left chest wall (the tumor was focally infiltrating the pectoralis muscle). We would like to show that a reconstruction of the breast in a young woman presenting with a bad prognosis can be carried out provided a well-organized team-work.


Intracochlear application of acetylcholine alters sound-induced mechanical events within the cochlear partition. Activation of olivocochlear (OC) efferent fibers has been suggested to alter micromechanical events occurring within the cochlear partition, possibly through an effect of the efferent neurotransmitter (acetylcholine; ACh) on outer hair cells (OHCs). Based on the widely-accepted assumption that otoacoustic emissions reflect OHC activity, we investigated the in vivo influence of ACh on OHCs by studying alterations in emission amplitude with local ACh application. Distortion product otoacoustic emissions (DPOAEs) were measured in anesthetized guinea pigs before, during, and after intracochlear application of ACh (250 microM) with the cholinesterase inhibitor, eserine (20 microM). Perfusion of ACh/eserine was associated with a desensitizing reduction in DPOAE amplitude of approximately 4.4 dB. This reduction was intensity-dependent, with greater and more consistent reductions observed for DPOAEs elicited by low- than by moderate-intensity primaries. The response reduction was not seen during consecutive ACh perfusions performed without an intervening artificial perilymph wash, and was effectively blocked in the presence of pharmacologic antagonists of OC efferent activity (curare, 50 microM; strychnine, 50 microM). Finally, a similar alteration in DPOAE amplitude was never seen during perfusion of the control (artificial perilymph) solution alone. It is argued that these results support the hypothesis that OC efferent activation can alter sound-induced cochlear mechanical events.


Managing geriatric chronic constipation. There is a relationship between low-fiber diet and chronic constipation, especially among inactive, geriatric nursing home and homebound patients. Increased bran fiber intake helps to promote normal bowel functioning among these patients.


Processing of pure-tone and FM stimuli in the auditory cortex of the FM bat, Myotis lucifugus. FM bats perceive their surroundings during echolocation by analyzing frequency-modulated (FM) acoustic signals. Results from this study indicate a cortical organization in Myotis lucifugus which is largely made up of neurons sensitive to FM sounds (FM-sensitive neurons). Three types of neurons were distinguished by their responses to pure-tone and FM stimuli: (1) Type I FM-sensitive units (83%), Type II FM-sensitive units (13%) and pure-tone sensitive units (4%). Type I FM-sensitive units responded to pure tones, but exhibited greater response magnitudes to FM stimuli when the best FM swept through the BF. An orderly frequency representation was found when the frequencies of pure tones essential for response (EPTs) in Type I units were mapped along the cortical surface. The EPTs for Type I neurons were usually found within the last millisecond of a downward FM sweep. As outlined by two neuronal network models, both the responses of Type I and II units could likely result from the convergence of excitatory and inhibitory lower level neurons with slightly differing BFs. Type II units were selective for an FM sweep and showed negligible to no response to pure-tone stimuli. Pure-tone sensitive units exhibited weak or no responses to FM stimuli. These neurons were clustered in a small area located rostrodorsal to the tonotopic zone and had significantly lower best frequencies than adjacent EPT frequencies of Type I FM-sensitive neurons.


Central auditory metabolic activity induced by intense noise exposure. Neural activity in the central auditory system was mapped by measuring 2-deoxyglucose (2-DG) uptake during a one hour exposure to a two-octave (1414-5656 Hz) band of noise. Gerbils were exposed to 100, 110 or 120 dB SPL, intensities which can produce only temporary (100 dB) or both temporary and permanent (120 dB) hearing loss. Exposure to 100 dB SPL evoked high levels of neural activity throughout responsive regions of auditory nuclei. At 110 dB SPL, a central region of low neural activity was surrounded by areas exhibiting increased activity. At 120 dB SPL, neural activity was low in almost all areas of auditory nuclei. To study the effects of permanent hearing loss on auditory neuronal activity, other animals were given 2-DG during exposure to 65 dB SPL broad band noise as a test stimulus, two months after exposure to the noise band at 110 dB SPL. Central auditory nuclei showed a tonotopic region of low neural activity corresponding to an approximately 3 kHz pure tone, surrounded by regions of evoked activity. The deficits in evoked metabolic activity observed both during and long after noise exposure appear to exceed those predicted from the degree of temporary and permanent threshold shift produced by the same noise exposures.


Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils. Changes in the integrity of cochlear ion transport systems with age were examined in gerbils raised for 5-38 months in a quiet environment. Ion transport function was assessed by light microscopic immunohistochemical staining for the enzyme, Na,K-ATPase and by measurement of the endocochlear potential (EP). Small foci of strial atrophy accompanied by loss of immunostaining for Na,K-ATPase were observed in the stria vascularis of the apical and basal turns as early as 5 months of age. Cochleas from 29-38 month-old gerbils showed a loss of immunostaining for Na,K-ATPase in the stria in most of the apical turn with the degeneration extending well into the middle turn in many of the oldest ears. The extent of strial atrophy and loss of immunoreactive Na,K-ATPase in the basal turn varied considerably among the oldest cochleas. Populations of lateral wall fibrocytes (type II fibrocytes) normally rich in Na,K-ATPase exhibited a corresponding decrease in enzyme content in regions of advanced strial atrophy. The volume of immunostained stria vascularis correlated well with the magnitude of the resting EP. The results demonstrate that lateral wall ion transport systems in the gerbil cochlea degenerate as a function of age. The findings also provide good evidence for a functional relationship between the stria vascularis and the Na,K-ATPase-rich type II fibrocytes in generating and maintaining the EP.


Luteal function and reproductive response in suckled beef cows after metestrus administration of a norgestomet implant and injection of estradiol valerate with various dosages of injectable norgestomet. In an experiment replicated over 2 yr, 149 suckled beef cows were administered Syncro-Mate-B (SMB), a 6-mg Norgestomet (NOR) ear implant (in situ 9 d) in conjunction with an i.m. injection of 5 mg of estradiol valerate (EV), and either 3.0, 4.5, or 6.0 mg of NOR, 2 d after estrus. All cows were artificially inseminated at 48 h (timed insemination; TI) after implant removal (IR) and cows were reinseminated at any estrus subsequent to 24 h of TI through 30 d. Blood samples collected before treatment, every 3 d through IR, and at TI were assayed for progesterone (P4). At TI, 44, 39, and 12% of cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively, had serum concentrations of P4 greater than 1 ng/mL (3.0 and 4.5 mg vs 6.0 mg, P less than .01). Fifty-eight, 63, and 84% of cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively, exhibited a synchronized (within 5 d of IR) estrus (3.0 and 4.5 mg vs 6.0 mg, P less than .05). Pregnancy rates for the 5-d synchronized period were 38, 45, and 66% for cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively (3.0 and 4.5 mg vs 6.0 mg, P less than .05). First-service pregnancy rates were 66, 71, and 79% for cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)


Passive immunization with antiserum to adrenocorticotropin increases weight gain in normal female rats. Passive immunization against ACTH was used to test the hypothesis that growth in female rats is constrained by physiological concentrations of glucocorticoids. When animals were stressed by 15-min exposure to ether before blood sampling by cardiac puncture, serum concentrations of corticosterone were lower (P less than .05) in immunized rats than in stressed controls. The maximum effect was apparent 2 h after injection of ACTH antiserum, and no effect was apparent 6 h after injection. To examine the effects of ACTH immunization on growth, rats received daily injections of either saline, sheep immunoglobulin G, or ACTH antiserum, 2 h before the afternoon peak in plasma concentrations of corticosterone. After 7 d of treatment, rats treated with ACTH antiserum had gained 37% more body weight than saline-injected controls, and this effect was accompanied by a 59% reduction in peak plasma concentrations of corticosterone. Immunoglobulin G purified from normal sheep serum had no effect on weight gain. It is concluded that growth rate in normal female rats can be stimulated through the suppression of adrenal activity.


Effect of dietary copper on intestinal mucosa enzyme activity, morphology, and turnover rates in weanling pigs. Twenty-four pigs from four litters weaned at 21 d of age (6.6 kg of BW) were used to evaluate the influence of 250 ppm of dietary Cu on intestinal mucosa glucose-6-phosphatase (GP), alkaline phosphatase (AP), and adenosine triphosphatase (ATPase) activity; mucosal morphology; and the turnover rate of the intestinal mucosa throughout the gastrointestinal tract. Pigs were allotted into four pens of six pigs each based on sex, litter, and weight. Pens were then assigned to one of two treatments: 1) corn-soybean meal-whey diet with no antimicrobials (CO), or 2) CO + 250 ppm of Cu. Pigs were fed twice daily an amount approximately equal to ad libitum intake for 14 d. On d 14, pigs were injected i.p. with [3H]thymidine (50 microCi/kg of BW) 10 h after the morning meal. One pig from each pen was euthanatized at 1, 6, 12, 20, 32, and 44 h postinjection, and intestinal tissue was collected from the duodenum, two jejunum sites (upper and lower), ileum, cecum, and colon. The activity of GP and AP in the lower jejunum tended to decrease in pigs fed Cu (P less than .11, P less than .08, respectively). The ATPase activity was not affected by treatment (P greater than .10). Crypt death, villus height, or epithelial cell size (P greater than .10) were not affected by feeding Cu. Migration rate of epithelial cells up the villus was also not affected by treatment (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of ruminal escape protein and fat on nitrogen utilization in lambs exposed to elevated ambient temperatures. Eight wether lambs (mean BW = 28.8 kg) with ruminal and abomasal cannulas were assigned to either thermally neutral or high ambient temperature treatments. Within each temperature, lambs were randomly allotted to dietary treatments consisting of a basal diet (60% corn and 24% cottonseed hulls) either with (high; 11.4% CP) or without (control; 10.1% CP) added ruminal escape CP as fish meal and with (high) or without (control) 5% added ruminally inert fat in a 2 x 2 factorial treatment arrangement using a Latin square design. Lambs were fed 606 g of DM/d in each period, which consisted of a 10-d adjustment followed by 6 d of sample collection. High temperature increased (P less than .05) respiration rate, evaporative water loss, and rectal temperature. When compared with controls, lambs fed high escape CP retained more N when exposed to high temperatures (2.8 vs 3.6 g of N/d) and less N at neutral temperatures (3.3 vs 3.1 g of N/d; temperature x escape CP; P less than .05). Retention of N was greater (P less than .05) in lambs fed high than in those fed control fat (3.8 vs 2.7 g/d). Lambs fed high vs control escape CP had greater abomasal feed N flow (percentage of intake) when fed high-fat diets (77.3 vs 56.1%) but similar dietary N flow when fed control fat diets (55.8 vs 54.3%; fat x escape CP; P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of supplemental protein percentage and feeding level on intake, ruminal fermentation, and digesta passage in beef steers fed prairie hay. Twelve ruminally cannulated steers (average initial BW 357 kg) were allotted to four treatments (three steers per treatment) in a replicated 4 x 4 Latin square design with 21-d periods (12 d for adaptation and 9 d for collection) to compare the effects of protein supplements that differed in percentage of CP and feeding level on low-quality forage utilization. Treatments were 1) control (C), ad libitum access to 5.6% CP prairie hay, 2) C +600 g of DM.steer-1.d-1 of a 43% CP supplement based on cottonseed meal (PS), 3) C + 1,200 g of DM.steer-1.d-1 of a 22% CP supplement based on corn grain and cottonseed meal (GS), and 4) C + 600 g of DM.steer-1.d-1 of a 22% CP supplement based on corn grain and cottonseed meal (LS). Ruminal total VFA concentrations were increased 8% (P less than .07) by PS vs GS 1 h after supplementation. Among supplemented steers, ruminal acetate (mol/100 mol) was decreased 1.2 mol/100 (P less than .03) by GS vs PS and LS; however, supplementation did not affect (P greater than .10) acetate proportions compared with C. Neither propionate nor butyrate was affected (P greater than .10) by supplementation, but among supplemented steers, butyrate proportions were 8% greater (P less than .03) for GS than for PS and 5% less (P less than .10) for LS than for the average of GS and PS. Ruminal pH did not differ (P greater than .10) among treatments. Ruminal ammonia concentrations were increased 1.4 to 4.8 mg/100 mL (P less than .07) by supplementation and typically were less for LS than for PS and GS at most sampling times. Prairie hay DMI (average = 16.3 g/kg BW) was not affected (P greater than .10) by supplementation. Fluid dilution rate was 8% faster (P less than .01) when steers were supplemented than when they were not fed supplement, and fluid dilution rate was increased 4% (P less than .04) by GS compared with PS. Particulate digesta passage rate was not affected (P greater than .10) by treatment, but total tract retention time was decreased (P less than .01) 10% by supplementation. Extent and rate of prairie hay NDF digestion in situ were not greatly affected by supplementation, but in situ disappearance of supplement N was 6 to 10 percentage units less (P less than .06) for GS than for PS and 2 to 6 percentage units less for LS than for the average of PS and GS supplements.(ABSTRACT TRUNCATED AT 400 WORDS)


Changes in monkey horizontal semicircular canal afferent responses after spaceflight. Extracellular responses from single horizontal semicircular canal afferents in two rhesus monkeys were studied after recovery from a 14-day biosatellite (COSMOS 2044) orbital spaceflight. On the 1st postflight day, the mean gain for 9 different horizontal canal afferents, tested using one or several different passive yaw rotation waveforms, was nearly twice that for 20 horizontal canal afferents similarly tested during preflight and postflight control studies. Adaptation of the afferent response to passive yaw rotation on the 1st postflight day was also greater. These results suggest that at least one component of the vestibular end organ (the semicircular canals) is transiently modified after exposure to 14 days of microgravity. It is unclear whether the changes are secondary to other effects of microgravity, such as calcium loss, or an adaptive response. If the response is adaptive, then this report is the first evidence that the response of the vestibular end organ may be modified (presumably by the central nervous system via efferent connections) after prolonged unusual vestibular stimulation. If this is the case, the sites of plasticity of vestibular responses may not be exclusively within central nervous system vestibular structures, as previously believed.


Altered actin and myosin expression in muscle during exposure to microgravity. The mechanism for cardiovascular deconditioning and skeletal muscle atrophy during microgravity is not known. The purpose of the present study was to determine whether a decrease in contractile protein gene expression in the muscle of rats occurred after 14 days of microgravity. No differences existed in the profile of myosin protein isoforms or beta-myosin heavy chain mRNA in hearts between the flight and synchronous control groups. On the other hand, differences in the expression of beta-myosin heavy chain mRNA relative to the 18S and 28S rRNA in the heart between flight and synchronous control groups were noted with a covariance mapping analysis. Both the vastus intermedius and lateral gastrocnemius muscles exhibited significant (P less than 0.05) decreases in skeletal alpha-actin mRNA per unit of extractable RNA in the flight group compared with the synchronous control group. However, no significant difference for skeletal alpha-actin mRNA occurred in the triceps brachii muscle between these groups. Cytochrome c mRNA per unit of extractable RNA decreased (P less than 0.05) only in the vastus intermedius but not in the lateral gastrocnemius or triceps brachii muscles. In summary, changes in the pretranslational regulation of contractile protein gene expression occur in both heart and skeletal muscle after 14 days of microgravity.


Proton transfer and energy coupling in the bacteriorhodopsin photocycle. A description of the rate constants and the energetics of the elementary reaction steps of the photocycle of bacteriorhodopsin has been helpful in understanding the mechanism of proton transport in this light-driven pump. The evidence suggests a single unbranched reaction sequence, BR-hv----K in equilibrium with L in equilibrium with M1----M2 in equilibrium with N in equilibrium with O----BR, where coupling to the proton-motive force is at the energetically and mechanistically important M1----M2 step. The consequences of site-specific mutations expressed homologously in Halobacterium halobium have revealed characteristics of the Schiff base deprotonation in the L----M1 reaction, the reorientation of the Schiff base from the extracellular to the cytoplasmic side in the M1----M2 reaction, and the reprotonation of the Schiff base in the M2----N reaction.


Photointermediates of visual pigments.

Photointermediates of visual pigments. Much progress has been made in recent years toward understanding the interactions between various proteins responsible for visual transduction which are initiated by an activated state of visual pigments. However, the changes which take place in the visual pigments themselves to convert them to the activated state are more poorly understood. Many spectroscopic techniques have been applied to this problem in recent years and considerable progress has been made. A major goal of these efforts is to understand at which stages protein change occurs and to characterize its structural features. In the visual system evidence is accumulating, for example, that chromophore independent protein change begins immediately prior to lumirhodopsin formation. Considerable insight has been gained recently into the early intermediates of visual transduction and the stage is set to achieve similar understanding of the later intermediates leading to rhodopsin's activated state.


The ligand-binding domain of rhodopsin and other G protein-linked receptors. Rhodopsin is a member of the very large family of G protein-linked receptors. The members of this family show clear signs of evolutionary relatedness, primarily in amino acid sequence homology, topographical structure of the proteins in the membrane, and the fact that all of the receptors function through the intermediary action of a GTP-binding regulatory protein or G protein. Recently, it has become clear that the structural similarity of these receptors extends well beyond the rather crude comparison of membrane topography. Reviewed here are several studies in which site-directed mutagenesis and active-site-directed reagents were used to show that the ligand-binding pockets of these receptors are highly similar. They are similar despite the fact that the structures of their various ligands are very different.


Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochrome c oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochrome c reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochrome c oxidoreductase is strongly stimulated by added CoQ3, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of the Km for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14 mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ9 and CoQ10.


Dual regulation of the n type K+ channel in Jurkat T lymphocytes by protein kinases A and C. We have measured the activity of the n type K+ channel present in human (Jurkat) T lymphocytes using the patch clamp technique in the whole-cell configuration. We report that protein kinase A (PKA) and protein kinase C (PKC) modulate, in a dual manner, the K+ conductance in these cells. Activation of PKA decreases the amplitude of the current, as previously reported (Bastin, B., Payet, M. D., and Dupuis, G. (1990) Cell. Immunol. 128, 385-399), and this is also the case for 12-O-tetradecanoylphorbol-13-acetate-dependent activation of PKC. In contrast, inhibitors of PKC (H7, staurosporine, polymixin B, and anti-PKC antibody) increase the current amplitude. Of importance, down-regulation of PKC or its inhibition prevented the PKA-dependent inhibition of the K+ channels. Addition of alkaline phosphatase via the patch pipette increased the K+ conductance under basal conditions and reversed the inhibition produced by PKA. The dual modulation of K+ channels in Jurkat T cells is in agreement with the presence of consensus sequences in the primary structure of the n type K+ channel.


The rod transducin alpha subunit amino terminus is heterogeneously fatty acylated. Rod transducin (Tr), a heterotrimeric GTP-binding protein composed of alpha, beta, and gamma subunits, couples photolysis of rhodopsin to the activation of cyclic GMP phosphodiesterase in the vertebrate visual signal transduction cascade. To determine if T alpha r is covalently modified, we analyzed tryptic fragments of bovine retinal T alpha r using electrospray mass spectrometry, liquid chromatography/mass spectrometry, tandem mass spectrometry, and gas chromatography. A novel heterogeneous fatty acylation was detected at the NH2 terminus. Four types of NH2-terminal tryptic fragments of T alpha r were isolated, and each contained either a lauroyl (C12:0), myristoyl (C14:0), (cis-delta 5)-tetradecaenoyl (C14:1) or (cis,cis-delta 5, delta 8)-tetradecadienoyl (C14:2) fatty acyl residue amide-linked to the NH2-terminal glycine residue. NH2-terminal fatty acylation does not anchor T alpha r permanently in the membrane, since T alpha r used in these experiments was eluted without detergent from rod outer segment membranes.


The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities. In 1989, Shier and Watt identified a gene which was predicted to encode a new member of the insulin receptor (IR) family, and they called it the insulin receptor-related receptor (IRR) (Shier, P., and Watt, V. M. (1989) J. Biol. Chem. 264, 14605-14608). However, the tissues expressing this receptor, its ligand binding specificity and its signaling capability have remained unknown. In the present studies we report Northern blot analyses and polymerase chain reaction data, which indicate that the IRR mRNA is expressed in a variety of tissues, including the human kidney, heart, skeletal muscle, liver, and pancreas. In order to examine the ligand(s) recognized by IRR, we constructed a chimeric receptor with the extracellular domain of the IR replaced with that of IRR. This chimera was found not to bind radioactively labeled insulin, insulin-like growth factor I (IGF-I), or IGF-II. These ligands and relaxin, the only other known member of the mammalian insulin family, also failed to stimulate the tyrosine kinase activity of this chimeric receptor. A second chimeric receptor with the extracellular domain of IR and the kinase domain of IRR was also constructed and utilized to study the signaling capabilities of the kinase domain of IRR. This chimera exhibited high affinity insulin binding and insulin-stimulated tyrosine kinase activity. The kinase domains of the IR and IRR were found capable of phosphorylating the same spectrum of exogenous and endogenous substrates. However, Chinese hamster ovary (CHO) cells stably overexpressing the kinase domain of IRR exhibited elevated basal thymidine incorporation and 2-deoxyglucose uptake compared with CHO cells and CHO cells overexpressing wild-type IR. We conclude that: 1) IRR is expressed in the human kidney, heart, skeletal muscle, liver, and pancreas, 2) IRR does not appear to be the receptor of any known member of the insulin family, and 3) the tyrosine kinase of IRR appears to be similar to that of IR in both the spectrum of substrates phosphorylated and the biological responses stimulated.


Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures. Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.


Molecular cloning and expression of a phosphoinositide-specific phospholipase C of Dictyostelium discoideum. A number of phosphoinositide-specific phospholipases C (PLC) of different species have recently been cloned. The predicted amino acid sequences of these isoforms contain two highly conserved domains. Here we report the identification of a PLC gene of Dictyostelium by using the polymerase chain reaction. Primers were designed coding for highly conserved amino acid regions located within one of the conserved domains of PLCs. Cloning and sequencing of the polymerase chain reaction product revealed one unique PLC-like sequence. This sequence was used to screen a library and isolate several overlapping cDNA clones. The complete cDNA was expressed in Dictyostelium cells resulting in increased basal levels of inositol 1,4,5-trisphosphate and enhanced PLC activity. The identified Dictyostelium PLC, DdPLC, encodes a protein with a calculated molecular mass of 91 kDa. The deduced amino acid sequence contains the two conserved domains found in other PLC isoforms, separated by a short variable region. The C-terminal part of the protein shows strong homology with the mammalian PLC-delta isoform. DdPLC is expressed at all stages of development, with an increase in transcription during starvation and in the culminating fruiting body.


Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9. Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.


High specific activity 125I- and 35S-labeled vasopressin analogues with high affinity for the V1 and V2 vasopressin isoreceptors. Since iodination of the tyrosine residue in the pressin ring of vasopressins abolishes binding to the V2 (renal) isoreceptor, the low specific activity tritiated vasopressins have been the only radioligands available for this receptor. Alternative vasopressin radioligands are described in the present study. N-tert-Butoxycarbonyl- (N-t-Boc) 125I-tyrosine or [35S]methionine were conjugated to the 8th amino acid of lysine- (LVP) or deamino-ornithine-vasopressin via active succinimidyl esters. Following the purification on C-18 reverse-phase high pressure liquid chromatography, t-Boc removal, and a second high pressure liquid chromatography purification, specific activities of 2200 and 1300 Ci/mmol were obtained for the 125I- and the 35S-labeled ligands, respectively. These vasopressin analogues, conjugated outside the pressin ring, were found to bind with high affinity to the V1A (vascular) and V2 vasopressin isoreceptors (Kd less than or equal to 10(-9) M) and to retain the full biological activity of intact vasopressin. The present study demonstrates the possibility of producing high specific activity radioligands with high affinity for the V1A and V2 vasopressin isoreceptors by conjugating labeled moieties to the 8th amino acid of vasopressin analogues. Since these new radioligands have specific activities much higher than the tritiated ligands (1300-2200 versus 10-30 Ci/mmol), they should provide considerable advantages in the future study of the physiology and biochemistry of the AVP receptors.


Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes. An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.


Site-specific and random fragmentation of Cu,Zn-superoxide dismutase by glycation reaction. Implication of reactive oxygen species. Site-specific and random fragmentation of human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was observed following the glycation reaction (the early stage of the Maillard reaction). The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro62 and His63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5 kDa) fragment. In the second step, random fragmentation occurred. The ESR spectrum of the glycated Cu,Zn-SOD suggested that reactive oxygen species was implicated in the both steps of fragmentation. The same fragmentations were seen upon exposure of the enzyme to an H2O2 bolus. Catalase completely blocked both steps of the fragmentation process, whereas EDTA blocked only the second step. Incubation with glucose resulted in a time-dependent release of Cu2+ from the Cu,Zn-SOD molecule. The released Cu2+ then likely participated in a Fenton's type of reaction to produce hydroxyl radical, which may cause the nonspecific fragmentation. Evidence that EDTA abolished only the second step of fragmentation induced by an H2O2 bolus supports this mechanism. This is the first report that a site-specific fragmentation of a protein is caused by reactive oxygen species formed by the Maillard reaction.


The phase behavior of cholesteryl esters in intracellular inclusions. Differential scanning calorimetry and polarizing light microscopy have been used to investigate kinetic and thermodynamic properties of the phase behavior of cholesteryl ester contained in Fu5AH rat hepatoma cells and J774 murine macrophages. These cultured cells store cholesteryl esters as cytoplasmic inclusions of approximately 1-micron diameter and thus are models of the foam cells characteristic of atherosclerotic plaque. Simple binary mixtures of cholesteryl palmitate and cholesteryl oleate, the predominant cholesteryl esters in cellular inclusions in both cell types serve as models to explain important aspects of the phase behavior of these inclusions. Although inclusions should exist as stable crystals at 37 degrees C under conditions of thermodynamic equilibrium, microscopic examination of cells indicates that inclusions exist as metastable liquid crystals at 37 degrees C for extended periods of time. Using an analytical model based on nucleation theory, we predict that the cholesteryl ester inclusions should be liquid-crystalline in the cytoplasm of living cells. This may not be true either for lysosomal cholesteryl ester or for extracellular cholesteryl ester present in advanced atherosclerotic plaque where fusion of droplets can enhance the possibility of crystallization. The enhanced metastability of the relatively fluid liquid-crystalline state in cellular inclusions should result in increased activity of the neutral cholesteryl ester hydrolase in living cells.


Identification of glycoinositol phospholipids in rat liver by reductive radiomethylation of amines but not in H4IIE hepatoma cells or isolated hepatocytes by biosynthetic labeling with glucosamine. The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.


Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein-terB complex of Escherichia coli. The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined. In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems. With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient. These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner. We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence. The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen. With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E. coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation. However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked. Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.


Molecular cloning of cDNA encoding a "63"-kDa calmodulin-stimulated phosphodiesterase from bovine brain. Partially degenerate oligonucleotides based on peptide sequence were used to isolate cDNA to a 63-kDa bovine brain calmodulin-stimulated phosphodiesterase (CaM-PDE) isozyme. A 412-base pair polymerase chain reaction fragment was obtained and used along with the oligonucleotides to isolate several cDNAs each encoding sequence identical to known peptide sequences from the 63-kDa CaM-PDE. The largest cDNA contained a full-length open reading frame (ORF) encoding a 534 amino acid, 61,005-dalton protein. It had 59% amino acid identity to the 61-kDa bovine brain CaM-PDE and included a carboxyl-terminal conserved domain containing the PDE catalytic domain consensus sequences. The NH2-terminal region fits the criteria for a calmodulin-binding domain. When its expression was driven by a cytomegalovirus promoter on a pCDM8 vector in COS-7 cells, the cDNA encoded a catalytically active, calmodulin-stimulated PDE. Northern analysis of RNA from several tissues with a probe containing much of the conserved PDE catalytic domain showed only a single band of 4.0 kilobases. Hybridization was seen in mRNA from several regions of the central nervous system with the greatest signal in basal ganglia. Strong signals also were seen in other tissues including kidney papilla and adrenal medulla. Antisense RNA probes were used in RNase-protection assays to look for evidence of multiple 63-kDa CaM-PDE transcripts. A catalytic domain probe was fully protected by RNA from cerebral cortex, basal ganglia, cerebellum, hippocampus, adrenal medulla, and kidney papilla. However, a probe to the NH2-terminal region was fully protected only by brain and adrenal medullary RNA indicating the likelihood of one or more isozyme(s) divergent in this region in the kidney papilla.


A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase. Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.


Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity. Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.


Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.


Organization, structure, chromosomal assignment, and expression of the gene encoding the human endothelin-A receptor. We have isolated and characterized the gene for the human endothelin-A receptor. Southern blot analyses demonstrated a single copy gene for the receptor. The gene spans more than 40 kilobases and contains eight exons and seven introns. Intron 1 exists in the 5'-noncoding region, and introns 2-7 occur in the coding region. The locations of introns 2-7 exist before or after the regions encoding the membrane-spanning domains. The transcription start site, determined by primer extension experiments, is 502 base pairs upstream of the methionine initiation codon. The 5'-flanking region lacks a typical TATA box but contains a potential SP-1-binding site 27 base pairs upstream of the transcription start site. Using human-rodent somatic hybrid cell DNA, the gene was assigned to human chromosome 4. Northern blot analyses revealed a 4.3-kilobase mRNA in a wide variety of human tissues, at the highest level in the aorta and at a substantial level in the cultured human mesangial cells. This is the first report of cloning of a gene for a member of the endothelin receptor family. The present study should give a clue to the discovery of possible disorders of the endothelin-A receptor, as well as facilitate the elucidation of the mechanisms by which the gene expression is regulated.


Histone shuttling by poly(ADP-ribosylation). We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(ADP-ribose) glycohydrolase, may cooperate to function as a histone shuttle mechanism on DNA. The mechanism involves four distinct reaction intermediates that were analyzed in a reconstituted in vitro system. In the first step, the enzyme poly(ADP-ribose) polymerase is activated in the presence of histone-DNA complexes and converts itself into a protein carrying multiple ADP-ribose polymers. These polymers attract histones that dissociate from the DNA as a histone-polymer-polymerase complex. The DNA assumes the electrophoretic mobility of free DNA and becomes susceptible to nuclease digestion (second step). In the third step, poly(ADP-ribose) glycohydrolase degrades ADP-ribose polymers and thereby eliminates the binding sites for histones. In the fourth step, histones reassociate with DNA, and the histone-DNA complexes exhibit the electrophoretic mobilities and nuclease susceptibilities of the original complexes prior to dissociation. Our results are compatible with the view that the poly(ADP-ribosylation) system acts as a catalyst of nucleosomal unfolding of chromatin in DNA excision repair.


Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity. The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.


Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain. Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.


Divalent cation-dependent stimulation of ligand binding to the 46-kDa mannose 6-phosphate receptor correlates with divalent cation-dependent tetramerization. The quaternary structure and binding activity of the murine 46-kDa mannose 6-phosphate receptor (46MPR) were studied in semi-intact murine cells that overexpress the murine receptor. Chemical cross-linking studies showed that the murine 46MPR exists in monomer, dimer, and tetramer forms in membranes of overexpressing murine cells. Treatment of permeabilized cells with Mn2+ increased the tetramer form of 46MPR, and this tetramerization was reversed by removal of Mn2+. Thus, the divalent cations affected the distribution of receptor among the three forms, favoring tetramerization at the expense of dimer and monomer. Low temperature (4 degrees C) also increases the fraction present as tetramer. The binding assay results show that Mn2+ is required for the 46MPR to achieve and retain the ability to bind ligand at 37 degrees C but not at 4 degrees C. Preincubation with Mn2+ produced a 3-fold increase in Man-6-P-specific binding of beta-glucuronidase which paralleled the 3-fold increase in tetramer seen during preincubation with Mn2+. The similarity of the effects of addition and removal of Mn2+ on enzyme binding to the effects of Mn2+ on favoring tetramer formation suggests that divalent cation-dependent tetramerization of the 46MPR contributes to the stimulation of ligand binding to the 46MPR by divalent cations.


Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells. Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.


The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase. The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.


The peroxisome proliferator-activated receptor mediates the induction of CYP4A6, a cytochrome P450 fatty acid omega-hydroxylase, by clofibric acid. Gene transfer experiments indicate that induction by the peroxisome proliferators, clofibric acid and WY-14,643, of luciferase expression driven by the promoter and 5'-flanking sequences of the rabbit cytochrome P450 4A6 gene (CYP4A6) is dependent on cotransfection of expression plasmids for the peroxisome proliferator-activated receptor, PPAR. Activation by PPAR is observed in the absence of the inducers. However, a mutant, PPAR-G (Glu282----Gly) activated luciferase expression only in the presence of peroxisome proliferators. Deletion analysis has localized a response element to a 34-base pair segment located 677 base pairs upstream of the CYP4A6 transcription start site that is similar to an element that regulates the response of the rat fatty acyl-CoA oxidase gene to peroxisome proliferators and that binds PPAR.


Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803. In certain cyanobacteria and algae, cytochrome c553 or plastocyanin can serve to carry electrons from the cytochrome bf complex to photosystem I. The availability of copper in the growth medium regulates which protein is present. To investigate copper induced control of gene expression we isolated these proteins from the cyanobacterium Synechocystis 6803. Using immunodetection and optical spectroscopy, the steady state levels of cytochrome c553 and plastocyanin were measured in cells grown at different copper concentrations. The results show that in cells grown in 20-30 nM copper, cytochrome c553 was present, whereas plastocyanin was not detected. The opposite behavior was observed in cells grown in the presence of 1 microM copper; plastocyanin was present, whereas cytochrome c553 could not be detected. Both proteins were present in cells grown in 0.3 microM copper. Northern analysis of total RNA, probed with a gene fragment for cytochrome c553 or the plastocyanin gene, showed that cells grown in the presence of 20-30 nM copper have message for cytochrome c553, but not for plastocyanin, whereas cells grown in 1 microM copper have message for plastocyanin, but not for cytochrome c553. These results demonstrate that copper regulates expression of both of the genes encoding cytochrome c553 and plastocyanin prior to translation in Synechocystis 6803.


Direct evidence for interaction between COOH-terminal regions of cytochrome b558 subunits and cytosolic 47-kDa protein during activation of an O(2-)-generating system in neutrophils. Cytochrome b558 in phagocytes is a transmembrane protein composed of large and small subunits and considered to play a key role in O2- generation during the respiratory burst. The COOH-terminal regions of the cytochrome subunits protrude to the cytoplasmic side and are assumed to be the sites for association with cytosolic components to form an active O(2-)-generating complex (Imajoh-Ohmi, S., Tokita, K., Ochiai, H., Nakamura, M., and Kanegasaki, S. (1992) J. Biol. Chem. 267, 180-184). We show here that two synthetic peptides corresponding to the COOH-terminal region of each subunit inhibit NADPH-dependent oxygen uptake induced by sodium dodecyl sulfate (SDS) in a cell-free system consisting of plasma membrane and cytosol. The inhibition was observed when either peptide was added to the system before, but not after, the activation with SDS suggesting that interaction between the COOH-terminal regions of the cytochrome subunits and cytosolic components is important for the assembly and the activity of the O(2-)-generating system. Using the cross-linking reagent dimethyl 3,3'-dithiobis-propionimidate, we found that the cytosolic 47-kDa protein, an essential component of the O(2-)-generating system, interacted with the synthetic peptides in the presence of SDS. In addition to the 47-kDa protein, a 17-kDa protein was found to be associated with the peptide corresponding to the COOH-terminal region of the small subunit. These results indicate that the cytosolic COOH-terminal regions of cytochrome b558 subunits are the binding sites for both the cytosolic 47-kDa protein and the 17-kDa protein and that the binding takes place during activation of the system.


Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein. A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.