Analysis of monoclonal rheumatoid factors obtained from the B-cell repertoire in rheumatoid arthritis.

We have sought to determine whether rheumatoid factors (RF) produced in rheumatoid arthritis (RA) were different from physiological RF produced in normal, healthy adults. RF-secreting clones were established following Epstein-Barr virus (EBV) stimulation of peripheral blood lymphocytes. Ten RF-secreting clones were established from seven RA patients and 16 from six healthy controls. All monoclonal RF (MRF), except two in each group, were monoreactive and ten of these were shown to have low to medium affinity for IgG,Fc, irrespective of their origin. A majority (74%) of the MRF bound to protein A, indicating that genes of the VHIII family were preferentially used for synthesizing these autoantibodies. The expression of cross-reactive idiotypes (CRI) by the MRF did not allow distinction between those derived from RA patients and controls. The VHI-associated CRI G8 and VHIII-associated CRID12 were expressed at low frequency in both panels of RF. These CRI have been shown to be expressed at high frequency in RF paraproteins. However, the idRQ idiotype was expressed within both panels of RF. A possible distinction between polyreactive and monoreactive MRF appeared to be light chain usage since all (four) polyreactive RF used lambda chains while the normal kappa/lambda ratio was observed for monoreactive RF. The frequency of EBV-activated cells secreting IgM bearing CRI or secreting RF was determined and showed that CRI expression occurred with a higher frequency than did RF, suggesting a dissociation between CRI expression and RF activity.


Interleukin-4 down-regulates Sendai virus-induced production of interferon-alpha and -beta in human peripheral blood monocytes in vitro.

Recombinant interleukin-4 (rIL-4) caused a 65-70% reduction of the interferon-alpha (IFN-alpha) and -beta production induced by Sendai virus (SV) in human peripheral blood monocytes in vitro. Significant inhibition was seen at concentrations as low as 0.1 ng/ml. The rIL-4 also reduced levels of IFN-alpha and -beta mRNA by 60% and 67%, respectively, as well as the frequency of IFN-alpha and -beta mRNA-containing cells by 65% and 54%, respectively. The frequency of IFN-alpha/beta mRNA-containing cells was inhibited by rIL-4 throughout the whole course of induction by SV. IL-4 caused a shift of the grain count distribution towards less heavily labelled cells, suggesting an inhibitory effect of rIL-4 on most IFN-alpha/beta mRNA-containing cells. Antibodies to rIL-4 did not influence the normal IFN-alpha/beta response induced by SV, but abolished the inhibitory effect of the rIL-4. When rIL-4 was added to cells 4 h after start of stimulation by SV, at which time much mRNA has accumulated but little IFN-alpha/beta has been secreted, no inhibition of the IFN-alpha/beta production by rIL-4 was seen. IFN-gamma had only a minor reversing effect on the rIL-4 inhibition, but if cells were precultivated in medium with or without IFN-gamma for 6 h before SV induction, rIL-4 paradoxically enhanced the IFN-alpha/beta response. Our results suggest that rIL-4 inhibits an early step of IFN-alpha/beta induction in monocytes, at the level either of transcription of IFN-alpha/beta genes or of the processing or stability of mRNA. The IL-4 effects may however depend on the state of activation of the monocytes.


Interferon-alpha but not -beta genes require de novo protein synthesis for efficient expression in human monocytes.

Monocytes produce interferon-alpha (IFN)-alpha) and -beta when human peripheral blood mononuclear cells (PBMCs) are stimulated in vitro by Sendai virus (SV). We found that about 70% of the IFN-producing cells (IPCs) expressed both IFN-alpha and -beta mRNA; the rest expressed only IFN-beta mRNA. In the presence of the protein synthesis inhibitor cycloheximide (CHX), the frequency of IFN-alpha mRNA-containing cells, measured after 6h, was decreased by 85-90%. Results of nuclear run-on transcription assays showed that CHX inhibited IFN-alpha gene expression. The frequency of IFN-beta mRNA-containing cells was not reduced by CHX. Actually, a threefold increase was observed at the lower CHX concentrations. Studies on the kinetics of IFN-alpha/beta mRNA induction showed that CHX accelerated the appearance of IFN-beta mRNA-containing cells, increased IFN-beta mRNA levels, and delayed the normally occurring post-inductional decrease of IFN-beta mRNA. Unexpectedly, an initially normal or even accelerated IFN-alpha mRNA response was seen in the presence of CHX during the first 3-4 h after SV stimulation. This occurred in a small proportion of the potential IPCs. However, CHX prevented the subsequent marked increase of IFN-alpha mRNA levels. Preincubation of PBMCs for 6 h in conditioned medium (CM) containing IFN and other cytokines prevented the CHX-mediated inhibition of IFN-alpha mRNA. Without preincubation this was not seen. The preincubation in CM caused an accelerated appearance of IFN-alpha mRNA, resembling that of IFN-beta mRNA. The results suggest that IFN-alpha and -beta genes are differentially regulated in the same monocytes, the former requiring de novo synthesis of intracellular protein(s) for efficient expression.


T-cell receptor beta-chain gene rearrangements of T-cell populations expanded from multiple sites of synovial tissue obtained from a patient with rheumatoid arthritis.

In this study T-cell receptor (TcR) beta-chain gene rearrangements of T-cell lines prepared from multiple sites (n = 92) of synovial tissue derived from both knees of a patient with rheumatoid arthritis were analysed. In the majority of T-cell lines, dominant TcR beta-chain gene rearrangements were detected, involving C beta 1 as well as C beta 2. The dominant rearrangement patterns of T-cell lines from different tissue fragments showed significant variability, but some of the DNA restriction fragments were shared by T-cell lines from multiple sites in both knees. The latter observation suggests that identical T-cell clones may be present at different sites in the synovial tissue and in different joints. However, since many T-cell lines yielded different rearrangement patterns, these data also indicate considerable heterogeneity of T cells in the joints. Apart from theoretical implications, this TcR heterogeneity of T cells within an individual patient also has practical consequences for studies on synovial T cells obtained by biopsy.


Prolonged elevation of intracellular cyclic AMP activates interleukin-1 production in human peripheral blood monocytes.

The capability of elevated intracellular cyclic AMP concentration to activate IL-1 gene expression and protein production was examined in human peripheral blood monocytes. In accordance with previous studies it was observed that the transiently elevated cyclic AMP (induced either with prostaglandin E2 or with the direct adenylate cyclase activator, forskolin) was not a sufficient signal to activate IL-1 production. However, if the degradation of cyclic AMP was inhibited with isobutyl-methyl-xanthine (IBMX), IL-1 production was strongly activated. This prostaglandin E2 plus IBMX effect could also be mimicked with high concentrations of the cell permeant structural cyclic AMP analogue, dibutyryl cyclic AMP. The cyclic AMP-induced IL-1 production differed in some aspects from the bacterial lipopolysaccharide-induced IL-1 production: (1) the kinetics of both IL-1 gene expression and protein production was much slower; (2) the IL-1 beta gene expression was superinducible by inhibiting the protein synthesis with cycloheximide. Thus these data suggest that prolonged elevation of cyclic AMP is alone a sufficient signal to activate IL-1 production.


Relationship between breast cancer and meningioma.

Several authors have suggested the existence of a relationship between breast cancer and meningioma occurring in the same patient. I describe four patients who had both of these neoplasms and present findings that dispute the implied relationship between the two.


Undifferentiated small cell carcinoma of the urinary bladder.

We have reported a case of small cell carcinoma of the urinary bladder in a 65-year-old white man with a smoking history. He had radical cystectomy, but died 6 months after initial diagnosis. The patient terminally had liver metastasis, confirmed by fine needle aspiration and radiographic evidence of intra-abdominal and right adrenal gland metastasis.


Primary squamous cell carcinoma of the breast.

We have described three patients with primary squamous cell carcinoma of the breast. All three have remained well after modified radical mastectomy. One patient received postoperative radiotherapy. Estrogen receptor status was determined by monoclonal antibody technology in two cases, and the results were negative.


Malignant fibrous histiocytoma arising from the superficial femoral artery.

Primary sarcomatous arterial tumors are rare. We have reported a case of malignant fibrous histiocytoma in an elderly woman successfully treated by resection of the involved femoral vessel and reconstruction with a PTFE graft. The long-term result in this patient is presently uncertain.


Regulation of pulmonary resistance during cardiopulmonary preservation.

To characterize the neural and vasoactive mediators of pulmonary vasoconstriction and determine whether the beneficial effect of isoproterenol could be mimicked by other agents that increase cyclic adenosine monophosphate, heart and lung organ blocks were harvested from calves and studied in a normothermic autoperfusion circuit. In the experimental protocol, measurements were obtained (A) after sternotomy but before autoperfusion (in vivo); (B) during stimulation of in vivo control with tert-butyl hydroperoxide (t-BuOOH), a lipid peroxide; (C) after cannulation and institution of in situ autoperfusion (innervated preparation); (D) following denervation and explanation (ex vivo); (E) during stimulation of ex vivo preparation with t-BuOOH; and (F,G) after administration of isoproterenol, aminophylline and prostaglandin E1 to the ex vivo preparation with (time G) and without (time F) stimulation using t-BuOOH. Plots of transpulmonary pressure gradient versus cardiac output were generated for each animal, and an index of pulmonary vascular resistance was obtained from the slope of the linear relationship. Blood samples were collected for measurement of thromboxane-B2, 6-keto-prostaglandin-F1 alpha, and complement activation products C3a and C5a. Pulmonary vasoconstriction occurred during in situ autoperfusion prior to denervation and increased further following denervation and ex vivo autoperfusion; vasoconstriction correlated with increased levels of circulating vasoactive mediators. The pulmonary vasoconstrictor response was greater in the denervated vascular bed compared with the innervated state. All agents reduced postexplant pulmonary vasoconstriction. We conclude that agents that increase cyclic AMP modulate the pulmonary vasoconstrictor response and thus may enhance lung preservation in the autoperfusion model as well as with other current preservation methods.


Liver transplantation for primary hepatic cancer.

Although early survival following transplantation for primary hepatic cancer is excellent, previously reported high recurrence rates have generally discouraged liver replacement for this indication. Since the inception of the Boston Center for Liver Transplantation (BCLT) in 1983, 33 of 383 (8.6%) liver allograft recipients have undergone orthotopic transplantation as definitive treatment for otherwise unresectable cancer. Diagnoses included hepatocellular carcinoma (HCCA) in 24 patients (73%), and cholangiocarcinoma (CHCA) in 9 patients (27%). Actuarial survival rates for patients with hepatocellular carcinoma were 71%, 56%, and 42% at 1, 2, and 3 years, respectively. The actuarial survival rates for patients with cholangiocarcinoma were 89% at 6 months, and 56% at 1, 2, and 3 years. Of the nine patients with cholangiocarcinoma, 56% (5/9) developed recurrent disease. Although this recurrence rate is disheartening, because of the lack of other morbidity, long-term survival in these patients is comparable to patients with HCCA. In contrast, recurrent hepatocellular carcinoma developed in 25% of recipients (5/20) who survived longer than 3 months posttransplantation. Other causes of death in patients with hepatocellular carcinoma included perioperative complications, 16.6% (4/24); sepsis, 8.3% (2/24); coronary artery disease, 4.2% (1/24); and lymphoma, 4.2% (1/24). Favorable prognostic factors included: primary tumor less than 3 cm in size and absence of associated cirrhosis. These results emphasize that orthotopic liver transplantation can provide a long-term cure for approximately 50% of patients whose primary hepatic malignancy is unresectable by conventional procedures.


Severe aseptic synovitis of the knee after biodegradable internal fixation. A case report.

Biodegradable rods made of polyglycolide (Dexon, Biofix) or lactide-glycolide copolymer (Vicryl) have been used for the past 5 years for internal fixation of a variety of fractures and osteotomies (Böstman et al. 1989, 1990b). Experience from using such rods for fixation of intraarticular osteochondral lesions seems to be less extensive. We report a case of severe synovial reaction to biodegradable rods used for fixation of osteochondritis dissecans of the knee and discuss possible reasons for the increased risk of foreign-body reactions when these rods are used intraarticularly.


Color Doppler sonography of hepatocellular carcinoma before and after treatment by transcatheter arterial embolization.

Color Doppler sonographic findings on 38 lesions in 31 patients who had primary hepatocellular carcinoma were evaluated before and after transcatheter arterial embolization and compared with dynamic CT and hepatic arteriographic findings. All lesions that were observed with dynamic CT or arteriography were correctly identified on color Doppler sonography. Peritumoral pulsatile flow was the predominant color Doppler flow seen in all lesions. Mixed pulsatile and continuous flow were noted in larger tumors and in tumors with a higher degree of vascularity, as determined by arteriography. Two weeks after treatment, color Doppler flow was still identified in 18 lesions (47%), corresponding to dynamic CT or arteriographic findings documenting residual tumor. Histopathologic examination, performed in 10 other lesions, showed that the tumor was completely necrotic in five. These five necrotic tumors were not visible on color Doppler flow images after treatment. Viable tumor was observed in the five remaining lesions, all of which were shown on color Doppler flow images after treatment. During the 6- to 16-month follow-up period, color Doppler flow images showed recurrence of 13 (50%) of 26 lesions, corresponding to tumor recurrence as shown by CT and arteriography. We conclude that color Doppler sonography is useful for imaging hepatocellular carcinoma, for evaluating residual tumor after treatment, and for imaging tumor recurrence during follow-up.


Renal handling of enalaprilat.

Most converting enzyme inhibitors share a predominantly renal dual elimination pathway consisting of glomerular filtration and tubular secretion. Since enalaprilat has two functional acidic groups, it is likely that it may be secreted via the proximal tubule organic acid system and, thus, its clearances would exceed that of glomerular filtration rate markers. We therefore examined the renal clearance of enalaprilat in normal volunteers and compared it with simultaneously measured inulin and creatinine clearances to explore the contribution of tubular secretion to the renal elimination of the drug. Twelve healthy male subjects with an age range of 24 to 58 years (mean +/- SE, 33.1 +/- 2.8) were studied. They had representative height (178.6 +/- 1.99 cm) and weight (73.3 +/- 2.1 kg) and had normal renal function as judged by blood urea nitrogen (BUN) (6 +/- 0.3 mmol/L [17 +/- 0.8 mg/dL]), plasma creatinine (88 +/- 3 mumol/L [1.0 +/- 0.03 mg/dL]), and creatinine clearance determined by a prestudy 24-hour urine collection (123.2 +/- 6.2 mL/min). Results are as follows: mean creatinine clearance, 2.12 mL/s (127 mL/min); mean inulin clearance, 119.1 ml/min mean creatinine clearance/inulin clearance, 1.07 mean enalaprilat protein binding, 37.9% unbound enalaprilat clearance, 222.4 ml/min; and the mean fractional enalaprilat clearances were: enalaprilat clearance/creatinine clearance, 1.72 (P less than 0.05, difference from 1.0); enalaprilat clearance/inulin clearance, 1.85, (P less than 0.05, difference from 1.0). Our results demonstrate that the clearance of free enalaprilat exceeds that of inulin and creatinine, suggesting that elimination of the drug proceeds through two complementary pathways, namely glomerular filtration and tubular secretion.


Correlation of the expression of Epstein-Barr virus latent membrane protein and in situ hybridization with biotinylated BamHI-W probes in Hodgkin's disease.

The detection of Epstein-Barr virus (EBV) nucleic acids by in situ hybridization (ISH) with biotinylated BamHI-W probes was correlated with the expressions of EBV latent membrane protein (LMP) and EB nuclear antigen 2 (EBNA2), in 107 cases of Hodgkin's disease (HD) of different immunomorphologic subtypes. Epstein-Barr virus nucleic acids were present and restricted to the pathogenic cells in 4 of 40 (10%) cases of nodular sclerosis (NS) and 33 of 55 (60%) cases of mixed cellularity (MC), but were undetectable in other subtypes. Of the 37 cases positive for EBV nucleic acids, 35 (95%) showed the expression of LMP. Epstein-Barr virus nucleic acids and LMP were restricted to Reed-Sternberg cells and variants. Only 1 case (MC) showed LMP expression in the absence of EBV detection. The correlation was strengthened by the finding of LMP expression at first diagnosis in 6/7 EBV positive cases at relapse (14-126 months) (5/5 EBV negative cases at relapse were LMP negative at first diagnosis). EBNA2 was absent in all 13 (NS, 2; MC, 11) EBV+ and LMP+ cases tested. Both LMP and EBNA2 were expressed in control EBV-positive tissues and cell lines. EBV serology in MC HD was indicative of latent EBV infection, but neither serology nor clinical parameters correlated with the presence or the absence of EBV, over a short-term follow-up (median, 20 months). The findings, although not proving EBV as the etiologic agent of HD, suggest that: 1) LMP expression alone may be adequate for identifying EBV-associated HD, 2) the MC subtype has a stronger relation with EBV presence, and 3) the regulation of EBV genes in HD is different from other EBV-associated disorders. The clinical implications still remain to be discovered.


Human papillomavirus 16 DNA immortalizes two types of normal human epithelial cells of the uterine cervix.

Premalignant cervical lesions occur at the squamo-columnar junction and in endocervical epithelium and squamous ectocervical epithelium, in descending order of frequency. However, previously only ectocervical cells have been clearly shown to be immortalized in vitro by the oncogenic human papillomaviruses (HPVs). This report describes the immortalization of normal human ecto- and endocervical epithelial cells by the intact HPV 16 genome. Ectocervical epithelial cells (HEC) became immortalized (HEC-16) without crisis while endocervical cells (HEN) were immortalized (HEN-16) after undergoing crisis. HEN-16 and HEC-16 contained integrated HPV 16 DNA, expressed E6 and E7 mRNA, and were aneuploid and nontumorigenic. They also expressed cytokeratins in a pattern similar to their distinct normal parental cells. These results suggest that both squamous and simple epithelial cells of uterine cervix are targets for immortalization by HPV 16.


Lack of ubiquitin immunoreactivities at both ends of neuropil threads. Possible bidirectional growth of neuropil threads.

Immunocytochemically, neuropil threads (curly fibers) were investigated in the Alzheimer's disease brain using a confocal laser scanning fluorescence microscope by double labeling with tau/ubiquitin antibodies. Ubiquitin immunoreactivities were found to be lacking at one or both ends in more than 40% of tau-positive threads. Immunoelectron microscopy showed that bundles of paired helical filaments, which constitute neuropil threads, were positive for ubiquitin around their midportions, but often negative at their ends. Since it is reasonable to postulate that tau deposition as paired helical filaments precedes ubiquitination, the aforementioned observation suggests that the ends of the threads are newly formed portions, and thus the threads are often growing bidirectionally in small neuronal processes.


Cellular localizations and processing of the two molecular forms of the Hodgkin-associated Ki-1 (CD30) antigen. The protein kinase Ki-1/57 occurs in the nucleus.

The Ki-1 antibody not only detects a Hodgkin-associated membrane molecule of 120 kd (Ki-1/120 = CD30), but also reacts with an independently synthesized molecule of 57 kd (Ki-1/57) that only occurs intracellularly. Hodgkin's disease-derived cell lines L428 and L540 contain both Ki-1-reactive antigens, whereas others, e.g., U266/Bl myeloma cells, only express the intracellular Ki-1/57. The present immunoelectronmicroscopic analysis detected the Ki-1/57 antigen of U266/Bl cells not only in the cytoplasm, but also in association with the nuclear envelope, chromatin structures, and nucleoli. This Ki-1/57-specific type of labeling also was observed in L428 and L540 cells that, in contrast to U266/Bl cells, showed an additional staining of cell membranes and cytoplasmic vesicles. These results were confirmed by two independent methods: 1) cytocentrifuge preparations of isolated nuclei of L540 cells showed a spotted Ki-1-specific labeling, 2) immunoprecipitations demonstrated that the Ki-1/57, but not the Ki-1/120 antigen, was transferred into the nuclei of L540 and U266/Bl cells, whereas the Ki-1/120 antigen with its 90-kd precursor remained in the non-nuclei fraction of L540 cells.


Human papillomavirus and the three group metaphase figure as markers of an increased risk for the development of cervical carcinoma.

In this study, the presence of atypical mitotic figures and human papilloma virus (HPV) genomes was related to the degree of cervical intraepithelial neoplasia (CIN) or microinvasive carcinoma (MIC) as found in 94 paraffin-embedded biopsies from cervical lesions. The results showed that the frequency of three group metaphase (TGM) figures, a special kind of atypical mitotic figure, as well as the presence of HPV 16 and 18 genomes increased with the degree of cervical intraepithelial neoplasia. TGM figures were observed in 24% of CIN2, up to 61% in CIN3 lesions, and in 83% of the microinvasive cervical carcinomas. HPV genomes were detected in 15% of CIN1, up to 75% in CIN3 lesions, and in 92% of the invasive carcinomas of the cervix. The combination of these two markers showed even a better association with a higher degree of cervical intraepithelial neoplasia. The results of these studies suggest that detection of particular HPV types, mainly HPV 16 and 18, and the presence of TGM figures can be considered as markers that indicate an increased risk for progression of cervical intraepithelial neoplasia to invasive carcinoma.


Effects of nedocromil sodium, cromolyn sodium, and a placebo in exercise-induced asthma.

The incidence and severity of exercise-induced asthma were determined in nineteen asthmatic patients who performed eight minutes of exercise following four treatments administered in a random order. The treatments were nedocromil sodium, cromolyn sodium, placebo, and no treatment. It was concluded that nedocromil sodium (8 mg) and cromolyn sodium (4 mg) provide equal protection against exercise-induced asthma.


Wheat-dependent exercise-induced anaphylaxis: inhibition by sodium bicarbonate.

We present a case of wheat-dependent exercise-induced anaphylaxis, in which pretreatment with sodium bicarbonate inhibited reappearance of anaphylactic symptoms following wheat and exercise provocation. Decrease in blood pH relative to elevation in plasma histamine levels was also inhibited. These results suggest possible efficacy of pretreatment with sodium bicarbonate as a preventive measure in patients with exercise-induced anaphylaxis.


Underutilization of Rh prophylaxis in the emergency department: a retrospective survey.

To determine the practice of emergency physicians with regard to the issue of Rh isoimmunization. A retrospective chart review. A university-affiliated tertiary care hospital emergency department. Pregnant women presenting with a risk factor for Rh sensitization. One hundred thirty-eight patient encounters were analyzed descriptively as to whether they were candidates for Rh immune prophylaxis, and if so, whether it was given. Most (68%) were hospitalized. Of those, all were Rh typed, but two patients were not given Rh immune globulin (RhIG) when indicated. Of those discharged from the ED, most (86%) were not Rh typed, and none was administered RhIG. This study demonstrates a need for increased attention to the potential for Rh isoimmunization in patients presenting to the ED.


Asymptomatic reactivation of herpes simplex virus in women after the first episode of genital herpes.

To determine frequency, anatomic site, and host factors associated with asymptomatic shedding of herpes simplex virus after initial episodes of genital herpes. Cohort study with follow-up for a median of 63 weeks. Referral clinic. Women (306) with first episode of herpes; 43 had primary herpes simplex virus type 1, and 227 and 36 had primary and nonprimary herpes simplex virus type 2, respectively. Cultures were obtained for herpes simplex virus every 4 to 6 weeks at times in which genital lesions and symptoms were not present. Asymptomatic shedding was detected among 11.9%, 18.3%, and 22.9% of women with primary herpes simplex virus type 1, primary HSV type 2, and nonprimary HSV type 2, respectively. Among patients with type 2 infection, previous type 1 antibody was associated with a lower rate of asymptomatic vulvar shedding. Asymptomatic cervical shedding was 3 times more frequent during the first three months after resolution of primary type 2 disease than during later time periods. In contrast, the rate of symptomatic recurrent herpes did not change over time. Asymptomatic genital herpes simplex type 2 is more common than type 1. Asymptomatic genital shedding occurs more often during the first 3 months after acquisition of primary type 2 disease than during later periods. Patients with HSV type 2 should be advised of this high early rate of asymptomatic shedding and of potential transmission to sexual partners.


Delay by internists in obtaining diagnostic biopsies in patients with suspected cancer.

To investigate the degree and type of delays in performing diagnostic biopsies in medical patients with suspected malignancy. Retrospective survey of clinical histories of patients referred between January 1985 and March 1989. Inner city teaching hospital internal medicine (non-oncologic) services. Patients with gastrointestinal and lung cancers, adenocarcinoma of unknown primary site, and lymphomas were referred as inpatients by internists. Two hundred fifty-five patients were eligible, and 177 were evaluable. The number, type, and results of tests done before and after biopsy were analyzed. In 67% of patients the biopsied lesion was detected by the second day of evaluation; however, there was an 8- to 10-day delay before a biopsy was done. This delay was consistent across the four malignancy groups studied. Although logistic and other unavoidable delays occurred in 40% of the cases, in 60% delays could only be attributed to continued, frequently low yield, noninvasive tests. An average of 3.3 tests were made per patient, with only 24% leading to a definitive biopsy. Because of the performance of many other tests, a substantial delay exists in proceeding to biopsy during the diagnosis of cancer by internists.


In situ hybridization and laryngeal papillomas.

The technique of in situ hybridization with biotin-labeled probes was applied to 20 patients with laryngeal papillomatosis (9 with adult-onset disease and 11 with juvenile-onset). Ten patients were noted for epithelial transformation of their laryngeal disease. All patients, regardless of their clinical outcome, tested positive for human papillomavirus group 6/11. The biotin method of in situ hybridization proved to be a sensitive method in this study, identifying the infecting viral group in 23 of 24 samples. This study also tested for group 16/18 and is the first to test for the presence of group 31/33/35 in the larynx. These viruses or members of related groups have been reported in malignancies of the head and neck as well as the uterine cervix. They have yet, however, to be identified in benign laryngeal papillomas or laryngeal papillomas that have undergone epithelial transformation.


Epstein-Barr virus-related persistent erythema multiforme in chronic fatigue syndrome.

Erythema multiforme (EM) has been rarely reported in Epstein-Barr virus (EBV)-associated diseases; this includes patients with chronic fatigue syndrome who have chronic or recurrent and disabling illness and an abnormal antibody reactivity to EBV. We describe a patient fulfilling the chronic fatigue syndrome diagnostic criteria who had developed an unusually persistent EM resistant to corticosteroids therapy. The EBV DNA was studied in skin EM lesions, throat washings, peripheral mononuclear cells, and plasma. The EBV antigens were studied in skin EM lesions and in mononuclear cells. The patient was followed up to 2 years. The patient had abnormal titers of antibodies against various EBV antigens and by immunofluorescence she disclosed the EBV nuclear antigen and the viral capsid antigen in the blood vessels of the affected skin. The dot blot hybridization assay detected viral DNA in throat washings and mononuclear cells, but not in plasma. The presence of the viral genomic content in lesional skin is suggested by the autoradiographic signal and by the difference from appropriate control specimens. Skin lesions and constitutional symptoms cleared after acyclovir sodium therapy and recurred after discontinuation of this therapy. This is the first EM case in which evidence of the EBV causal role has been provided. The association with chronic fatigue syndrome suggests the EBV role in selected cases of this syndrome.


Serum angiotensin-converting enzyme level is elevated in patients with human immunodeficiency virus infection.

Serum angiotensin-converting enzyme (ACE) level is elevated in a number of disease states and medical conditions. Based on review of the medical literature, we postulated that serum ACE level elevation may occur during infection with the human immunodeficiency virus (HIV). In a prospective, controlled study, serum ACE levels of HIV-positive patients were compared with those of an HIV-negative control population. Serum ACE levels were significantly elevated in patients with the acquired immunodeficiency syndrome (55.4 +/- 11.4 U/L) and in patients with an intermediate stage of HIV infection (57.2 +/- 25.3 U/L) when compared with levels in controls (31.9 +/- 14.0 U/L). Elevated serum ACE levels occur in HIV infection and may be an important marker for this disease.


Angiotensin-1-converting enzyme activity as index of pulmonary damage in thermal injury with or without smoke inhalation.

Serum angiotensin-converting enzyme (ACE) activity, plasma renin activity (PRA), and serum aldosterone levels were measured up to four weeks in a population of adults exposed to thermal injury, with or without concomitant exposure to smoke inhalation. In 10 patients, plasma levels of angiotensin-2 and ACE activity in bronchial lavage were also evaluated. Patients with severe burn injury had a significant decline of serum ACE activity while the concentrations of aldosterone and PRA were markedly elevated. Smoke inhalation seemed to counterbalance the decline of serum ACE activity, and, in the last group of patients, ACE concentrations were higher than those recorded in patients suffering only from cutaneous burn. The ACE activity was evidenced in bronchial lavage of patients exposed to smoke inhalation with the highest values present in the first day after the injury. The same patients had also very elevated levels of plasma angiotensin 2. In conclusion, serum ACE activity decreases in burn patients according to the severity of the cutaneous burn; smoke inhalation influences serum levels of the enzyme with concentration values opposite to the low ones present in cutaneous burn. Finally, the enzyme activity has an independent pattern from that of the other components of the renin angiotensin aldosterone system. The evaluation of ACE activity may be a marker of pulmonary damage in smoke inhalation.


APUDomas: acute complications and their medical management.

APUDomas are rare tumours originating from a variety of endocrine cells localized in different organs. Acute complications from APUDomas usually result from the increased biosynthesis and release of bioactive amines or polypeptide hormones by the tumour. Less frequently, bleeding or compression by the tumour can occur requiring emergency surgery. Increased gastrin production by gastrinomas is the cause of ZES (peptic ulceration and diarrhoea) by gastrin effects on gastric acid secretion. Volume depletion, hypokalaemia, severe bleeding, duodenal perforation, oesophageal stricture and pyloric stenosis are the most dramatic complications. Treatment of these complications and their prevention has been facilitated by the availability of antagonists to H2 receptors and H(+)-K+ proton pump. These medications should control acid output in every patient with ZES. Frequent manifestations of carcinoid tumours, VIPomas and medullary thyroid carcinomas are flushing and diarrhoea. Octreotide, a long-acting somatostatin analogue, has markedly changed the management of these patients, their symptoms decreasing in severity or disappearing in most cases. Octreotide has also been used with success in the prevention and treatment of the carcinoid crisis, a dreaded complication of carcinoid tumours. A better understanding of the pathophysiology of APUDomas has enabled new treatment designs which have considerably ameliorated the quality of life of patients affected by these tumours; efforts must be continued to affect their life expectancy.


Endotoxin-inducible cytotoxicity in liver cell cultures--II. Demonstration of endotoxin-tolerance.

Endotoxins from gram negative bacteria, central mediators of septic shock, share the characteristic property of inducing tolerance against their own action. This work investigates whether a corresponding ex-vivo tolerance can be observed in a cellular system with endotoxin-inducible hepatocytoxicity. The following experimental approaches were chosen in order to induce an endotoxin-unresponsive state prior to cell preparation: (1) pretreatment of rats with endotoxin, (2) partial hepatectomy, (3) use of neonatal rats and (4) pretreatment of rats with silica. An in-vivo protection against endotoxin-induced liver injury was obtained by all of these four measures: cells prepared from these groups of animals showed greatly diminished sensitivity towards endotoxin-induced hepatocytotoxicity in vitro. The suppressed endotoxin sensitivity after silica pretreatment was partially restored in vitro by the addition of native Kupffer cells (KC). Isolated KC of all but the endotoxin-pretreated animals secreted tumor necrosis factor-alpha in response to endotoxin. It is concluded that different types of tolerance can be distinguished: (a) impairment of macrophage functions (silica pretreatment), (b) hepatocyte unresponsiveness (neonatal rats and hepatectomy) and (c) impaired macrophage function combined with hepatocyte unresponsiveness (endotoxin-pretreated rats).


Effects of calcium and calcium-channel blocker methoxyverapamil on the beta-adrenoceptors in myocardial cells in vitro.

The possible relationship between methoxyverapamil (D600) as a calcium-channel blocker and the beta-adrenoceptors was investigated on heart cells grown in culture, using [3H]CGP-12177 as a radioligand. Treatment with D600 (20 micrograms/mL) for 24 hr caused a decrease of 30% in the [3H]CGP-12177 binding sites. Scatchard analysis showed that the Bmax is similar in control and D600-treated cells, but the Kd in D600-treated cells increases. The effect of D600 on the isoproterenol-induced adenylate cyclase activation was examined and it was found that the D600 prevented the increase in cAMP obtained by isoproterenol treatment. These results indicate that the action of D600 on the beta-adrenoceptors is a competitive inhibition of the [3H]CGP-12177 binding sites. We investigated the effect of Ca2+ in the growth medium on the level of beta-adrenoceptors. Heart cells grown for 24 hr in Ca(2+)-free medium showed a decrease of 36% in the [3H]CGP-12177 binding sites without changing the dissociation constant. This decrease is probably a result of reduction in synthesis of the receptors. The level of receptors returned to control values following replenishment with normal growth medium. These results show that calcium is essential for the development of the beta-adrenoceptors in heart cells in vitro.


Evidence that the loss of rat liver cytochrome P450 in vitro is not solely associated with the use of collagenase, the loss of cell-cell contacts and/or the absence of an extracellular matrix.

Two methods avoiding the widespread technique of collagenase perfusion have been employed to study the regulation of total cytochrome P450 content in rat hepatocyte culture. One technique required the perfusion of the liver with the chelating agent EDTA to dissociate the parenchymal cells prior to culture. Over a period of 48 hr, cultured hepatocytes isolated by EDTA perfusion showed comparable losses of cytochrome P450 as cells isolated by perfusion with collagenase. The second technique involved the culture of 210-240 microns thick "precision cut" liver slices. The results presented here indicate that the liver slices remain viable for 24 hr of culture, but that liver slices also lose their cytochrome P450 content at a comparable rate to collagenase prepared cells in culture. Collectively the results suggest that there is not a direct causal relationship between the loss of cytochrome P450 and one or a combination of the use of collagenase; the loss of cell-cell contacts and the absence of an extracellular matrix.


Superoxide, hydroxyl and fatty acid radical scavenging by aminosalicylates. Direct evaluation with electron spin resonance spectroscopy.

Reactive oxygen radicals such as superoxide and hydroxyl radicals, as well as intermediate unsaturated fatty acid radicals, have been proposed as playing an important role in various diseases including inflammatory bowel disease (IBD). In this study we evaluated radical scavenger properties of aminosalicylates used in the therapy of IBD using spin trapped electron spin resonance spectroscopy. 5-Aminosalicylic acid (5-ASA), 4-aminosalicylic acid and olsalazine had superoxide radical scavenger properties (IC50 = 0.4, 0.4 and 1.0 mM, respectively). 5-ASA and benzalazine also inhibited hydroxyl radicals (IC50 = 6.5 mM). Fatty acid radicals were not inhibited by aminosalicylates. Our results support the hypothesis that therapeutically active compounds may be oxygen radical scavengers and that fatty acid radical scavenging has to be performed by drugs other than aminosalicylates.


Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets.

Dicentrine is an antiplatelet agent isolated from the Chinese herb Lindera megaphylla. We examined the in vitro effects of dicentrine on various aspects of platelet reactivity. Dicentrine inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA), collagen, ADP, platelet-activating factor (PAF), thrombin and U46619. Dicentrine also inhibited the thromboxane B2 formation caused by AA, collagen and thrombin in washed intact platelets or that induced by AA in lysed platelet homogenate, while prostaglandin D2 formation caused by AA was not increased. The generation of inositol monophosphates (in the presence of indomethacin) caused by thrombin, collagen and PAF was not suppressed significantly, nor did dicentrine suppress fibrinogen-induced aggregation of elastase-treated platelets. Dicentrine inhibited the intracellular Ca2+ increase in quin-2/AM-loaded platelets caused by thrombin, PAF, collagen and AA. The cyclic AMP level was elevated by dicentrine in a concentration-dependent manner. These data indicate that the inhibitory effect of dicentrine on platelet aggregation and ATP release was due to the inhibition of thromboxane formation and the elevation of the level of cyclic AMP.


Components of intrinsic drug resistance in the rat hepatoma.

A carcinogen-transformed rat hepatoma cell line (Reuber H-35) was utilized as a model system for investigation of the biochemical factors which may limit the effectiveness of chemotherapy in intrinsically resistant tumors such as hepatocellular carcinoma. Northern blotting demonstrated expression of mRNA coding for the P-170 membrane-glycoprotein associated with the multi-drug resistance phenotype, while Western blotting identified the P-170 glycoprotein in the hepatoma cell membrane. Consistent with these observations, tumor cell sensitivity to the vinca alkaloids, vincristine and vinblastine, to the anthracycline antibiotics, Adriamycin and daunorubicin, and to the demethylepipodophyllotoxin derivative, VM-26, was enhanced by continuous incubation in the presence of the calcium channel antagonist, verapamil. Verapamil produced a minimal change in cell sensitivity to the demethylepipodophyllotoxin derivative, VP-16, and to the aminoacridine, m-AMSA. Relatively high detoxification potential via the glutathione metabolic pathway was also observed in the hepatoma cell. The capacity of topoisomerase II in nuclear extracts from the hepatoma cell to mediate cleavable complex formation stimulated by VM-26, VP-16 and m-AMSA appeared to be at least comparable to, if not greater than that from drug-sensitive HL-60 cells, suggesting that drug resistance may not occur at the level of this enzyme. Consistent with findings in a number of tumor cell lines resistant to antineoplastic drugs, the antiproliferative activity of the topoisomerase II inhibitors VM-26, VP-16 and m-AMSA appeared to be dissociable from the induction of DNA strand breaks, suggesting that such lesions in DNA may fail to fully account for the antiproliferative activity of these agents in the hepatoma cell.


One-electron reductive bioactivation of 2,3,5,6-tetramethylbenzoquinone by cytochrome P450.

Bioreductive activation of quinones in mammalian liver has generally been attributed to NADPH-cytochrome P450 reductase. However, in view of the 20-30-fold molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase on the endoplasmic reticulum of the rat liver cell and the capability of cytochrome P450 to bind and reduce xenobiotics, it was considered of interest to investigate the possible role of cytochrome P450 in the bioreduction of quinones. In the present study, 2,3,5,6-tetramethyl-1,4-benzoquinone (TMQ) was chosen as a model quinone. First, TMQ was found to bind at the metabolic active site of phenobarbital (PB)-inducible cytochrome P450s of rat liver microsomes, indicating that TMQ is a potential substrate for cytochrome P450-mediated biotransformation. Second, with electron spin resonance, one-electron reduction of TMQ to a semiquinone free radical (TMSQ) was found to occur in these microsomal fractions. SK&F 525-A, a well-known inhibitor of cytochrome P450, strongly inhibited TMSQ formation in these subcellular fractions without affecting NADPH-cytochrome P450 reductase activity. One-electron reductive bioactivation of TMQ was further investigated with purified NADPH-cytochrome P450 reductase alone and in reconstituted systems of purified cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase. As measured by ESR, purified cytochrome P450-IIB1 in the presence of NADPH-cytochrome P450 reductase was able to reduce TMQ to TMSQ at a much greater rate than in the presence of NADPH-cytochrome P450 reductase alone. Reduction of TMQ was also investigated by measuring the initial rate of NADPH oxidation by TMQ under anaerobic conditions. Inhibitors of cytochrome P450, namely SK&F 525-A and antibodies against PB-inducible cytochrome P450s, caused a substantial decrease in reductive metabolism in PB-treated microsomes. These antibodies were also effective in the inhibition of TMQ-induced NADPH oxidation in a complete reconstituted system of equimolar concentrations of cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase, indicating that the reaction was specific for cytochrome P450-IIB1. Finally, initial rates of NADPH oxidation were determined in reconstituted systems containing varying amounts of NADPH-cytochrome P450 reductase and cytochrome P450-IIB1 to determine the contribution of either enzyme in the reduction of TMQ. As expected, NADPH-cytochrome P450 reductase was able to reduce TMQ to a small extent. However, reconstitution in the presence of increasing amounts of cytochrome P450-IIB1 (relative to NADPH-cytochrome P450 reductase) resulted in increasing rates of TMQ-induced NADPH oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)


Guinea pig whole blood 5-lipoxygenase assay: utility in the assessment of potential 5-lipoxygenase inhibitors.

Guinea pigs are widely used in the study of the role of leukotrienes in airway pathophysiology. Extensive research efforts have utilized this species in the development of potential therapeutic agents associated with inhibition of leukotriene production (e.g. 5-lipoxygenase inhibitors and 5-lipoxygenase-activating protein antagonists) for the treatment of acute bronchospasm in asthma. We now report, for the first time, an ex vivo whole blood 5-lipoxygenase assay in guinea pigs which should prove useful in the future development of leukotriene biosynthesis inhibitors. Addition of 150 microM arachidonic acid (AA) to heparinized whole blood for 5 min prior to the stimulation with 20 micrograms/mL A23187 resulted in a 10-fold increase in leukotriene B4 (LTB4; 11.36 +/- 1.55 ng/mL) above basal (0.96 +/- 0.29 ng/mL) within 10 min. To further validate the utility of the assay, we utilized the 5-lipoxygenase inhibitor BW A4C. Pretreatment of guinea pig whole blood with BW A4C in vitro prior to stimulation resulted in a concentration-dependent inhibition of LTB4 production (IC50 = 229 nM), whereas thromboxane B2 (TXB2) production was unaffected. Likewise, when BW A4C was administered to guinea pigs intravenously (3 mg/kg), we observed a rapid and marked (approximately 90%) reduction in whole blood LTB4 production which returned to control (pre-drug values) by 5 hr. In contrast, TXB2 production was unaffected over the same experimental time period. In summary, we have described a whole blood assay which can discriminate 5-lipoxygenase inhibitors both in vitro and in vivo. Furthermore, this assay system will be of use in determining the potency, efficacy, selectivity, and pharmacodynamic properties of 5-lipoxygenase inhibitors in guinea pigs.


Electrodiagnostic characteristics of Wegener's granulomatosis-associated peripheral neuropathy.

Wegener's granulomatosis is a multisystem disease distinguished by a triad of necrotizing granulomatous vasculitis involving the upper and lower respiratory tracts, glomerulonephritis and systemic, small vessel vasculitis. The latter can cause neurologic manifestations when the vasa nervorum are affected. A 53-yr-old male presented with a 3-mo history of chronic nasal congestion, arthralgias, pruritic maculopapular eruption, epistaxis and lower extremity weakness. Subsequent lung and chest wall biopsies confirmed diagnosis of Wegener's granulomatosis. Summary of electrodiagnostic data obtained on initial presentation and comparison with later study indicated a sensorimotor polyneuropathy with wide-spread axonal involvement noted particularly in the distal lower extremity musculature. Electrodiagnostic documentation of rapid progression proved useful in directing alteration of immunosuppressive therapy, with favorable clinical and functional outcome. We believe this is the second case presented of a patient with documented Wegener's granulomatosis and overt clinical evidence of poly-neuropathy in whom both electroneurographic and electromyographic studies are described. Electrodiagnostic results are presented with discussion of pertinent literature.


Primary structure and expression of the gamma 2 subunit of the glutamate receptor channel selective for kainate.

The presence and primary structure of a novel subunit of the mouse glutamate receptor channel, designated as gamma 2, have been revealed by cloning and sequencing the cDNA. The gamma 2 subunit has structural characteristics common to the neurotransmitter-gated ion channel family and shares a high amino acid sequence identity with the rat KA-1 subunit, thus constituting the gamma subfamily of the glutamate receptor channel. Expression of the gamma 2 subunit together with the beta 2 subunit in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.


Sequence alignment of the G-protein coupled receptor superfamily.

The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.


Cloning and characterization of the human insulin-like growth factor-I receptor gene 5'-flanking region.

The insulin-like growth factor-I receptor (IGFIR) is a membrane-bound glycoprotein that mediates the action of insulin-like growth factors. The cDNAs for the human IGFIR have been cloned and expressed, but the structures of the gene and its promoter have not been elucidated. In this study, we isolated an IGFIR promoter clone from a human chromosome 15 library. This clone contained the promoter, first exon, and a portion of the first intron. Sequence analysis of the 5' region that contained the promoter revealed that it lacked both TATA and CAAT boxes. The promoter contained binding sites for the transcription factors Sp1, AP-2, and the epidermal growth factor receptor transcription factor (ETF). Primer extension analysis of IGFIR mRNA indicated the presence of a single transcription start site 1,012 bp upstream from the ATG. When the putative promoter was ligated into a promoterless CAT vector and transfected mto HEPG2 cells, CAT activity was expressed, indicating that promoter activity was contained in this fragment. Other constructs containing the promoter and portions of the 5' untranslated region were used in transfection studies, and indicated that the 5' untranslated regions may play a role in promoter activity. Comparison of the human IGFIR promoter with that of the rat IGFIR promoter revealed significant sequence homology. Comparison of the IGFIR promoter with that of the human insulin receptor (IR) revealed structural similarities, although the arrangement of promoter elements differed.


The cloned promoter of the human DNA beta-polymerase gene contains a cAMP response element functional in HeLa cells.

The mammalian DNA beta-polymerase (beta-pol) gene is constitutively expressed in cultured cells as a function of growth stage and DNA replication, but is expressed in rodents in a tissue-specific fashion. As revealed by transient expression experiments with wild-type and mutated beta-pol promoter fusion genes, the cloned human beta-pol promoter is transcriptionally regulated by signals acting through the single palindromic sequence (GT-GACGTCAC) known as an ATF/CRE-binding site centered at position -45 in the core promoter. Although the mere presence of the ATF/CRE palindromic sequence in a promoter does not always confer cAMP responsiveness or protein binding over and around the ATF/CRE sequence, we find that agents that increase cAMP levels (forskolin and IBMX) in HeLa cells activate the beta-pol promoter; activation also can be observed by coexpression of the protein kinase A catalytic subunit. Experiments with mutagenized beta-pol promoters indicate that the ATF/CRE-binding site mediates these effects. Thus, the ATF/CRE-binding site in the context of this TATA-less constitutive promoter is able to respond to the kinase A signal transduction pathway.


Possible role during exocytosis of a Ca(2+)-activated channel in neurohypophysial granules.

Ion channels from bovine neurohypophysial granules were incorporated into artificial lipid bilayers. The larger amplitude channel is permeable to cations and exhibits multiple conductances. The channel opens only in the presence of free Ca2+, but is inhibited by relatively high Ca2+ concentrations. Release of vasopressin from permeabilized neurohypophysial terminals also shows a similar biphasic dependence on Ca2+. Release is selectively inhibited by low concentrations of the long-chain alcohol octanol, but not by high concentrations of ethanol, as is the neurosecretory granule Ca(2+)-activated cation channel. Furthermore, Ca(2+)-evoked release and channel activity are both inhibited by the long-chain tetraethylammonium analogs decamethonium and decyl-triethyl ammonium bromide. The close correlation between channel and release properties lead us to conclude that the Ca(2+)-activated channel is involved in peptide secretion.


Nicotinic receptors that bind alpha-bungarotoxin on neurons raise intracellular free Ca2+.

Many populations of vertebrate neurons have a membrane component that binds alpha-bungarotoxin and cholinergic ligands. Despite the abundance of this component and its similarities to nicotinic receptors, its function has remained controversial. Using a fluorescence assay, we show here that activation of the component elevates the intracellular concentration of free Ca2+, demonstrating a receptor function for the toxin-binding component. Whole-cell voltage-clamp and intracellular recordings did not detect a significant current resulting from receptor activation, possibly because the currents were small or the receptors rapidly desensitized. The rise in intracellular free Ca2+ caused by the receptor was prevented by Ca2+ channel blockers. This suggests a signaling cascade likely to have important regulatory consequences for the neuron.


Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).


A regulatory subunit of the cAMP-dependent protein kinase down-regulated in aplysia sensory neurons during long-term sensitization.

Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation. To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI. Immunoblots with antisera raised against the R protein overexpressed in E. coli show that the diminished binding activity, which occurs in long-term facilitation, results from coordinate loss of R protein isoforms. No change was detected in the amount of transcripts for R subunits, suggesting that the down-regulation results from enhanced proteolytic turnover.


Differential expression of glutamate receptor genes (GluR1-5) in the rat retina.

The recent isolation of at least five different cDNAs encoding functional subunits of glutamate receptors (GluR1 to GluR5) has revealed a diversity whose function is not understood. To learn more about how these different receptor subunits are used in the brain, we undertook an in situ hybridization study of the retina to define how the different glutamate receptor genes are expressed. We chose the retina because the glutamate sensitivities of its different cell types have been characterized, and these different neurons reside in different laminae. Hybridization of [35S]UTP-labeled cRNA probes with transverse sections and freshly dissociated cells reveals that all five receptor subunits are expressed in the retina. Hybridization signal is detected in different, but overlapping, sets of cells in the retina. GluR1, GluR2, and GluR5 are expressed by many somata, and GluR4 by a few, in the outer third of the inner nuclear layer, where the horizontal cells reside. Transcripts for GluR1, GluR2, and GluR5 are found in the somata within the middle third of the inner nuclear layer, which is where the bipolar cell somata are located, and GluR2 probes label freshly dissociated rod bipolar cells. All of the probes produce labeling over the cells at the inner edge of the inner nuclear layer, which are probably amacrine cells, as well as over the cell bodies in the ganglion cell layer.


Localization of GABAA receptor subtypes in the tiger salamander retina.

Dry autoradiography was used to determine the distribution of GABAA binding sites in tiger salamander retina. High-affinity binding of [3H]-flunitrazepam [( 3H]-FNZ) was used to localize benzodiazepine receptors (BZR) and [3H]-muscimol was used to localize the GABAA recognition site. Specific [3H]-FNZ binding was present only in the inner retina, primarily in the inner plexiform layer (IPL). Co-incubation with GABA enhanced [3H]-FNZ binding by 20-50%. [3H]-muscimol binding was found throughout the IPL and in the outer plexiform layer (OPL). Mouse monoclonal antibodies 62-3G1 and BD-17, that recognize the GABAA beta 2, beta 3 polypeptides, and BD-24, that recognizes the GABAA alpha 1 polypeptide, did not label either the OPL or IPL, despite numerous variations in the fixation and immunoprocessing methods. GABAA receptor location, as revealed by [3H]-muscimol binding, matches the distribution of presumed GABAergic terminals in the OPL and IPL. We suggest that there are at least two subtypes of GABAA receptor in the tiger salamander retina: one type is present only in the inner retina, primarily in the IPL and is functionally coupled to BZRs; the other type is located in the OPL and is not coupled to the BZRs. Furthermore, GABAA receptors in the tiger salamander retina appear to be of a different epitope than GABAA receptors in numerous other preparations that are recognized by mAbs 62-3G1, BD-17, and BD-24.


Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex.

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.


Rates of interactions of superoxide with vitamin E, vitamin C and related compounds as measured by chemiluminescence.

The rate constants for the interactions of superoxide with vitamin E (alpha-tocopherol), vitamin C (ascorbic acid) and their related compounds have been measured by a chemiluminescence method. A strong chemiluminescence of a constant intensity was observed when xanthine oxidase was added to an aqueous solution of hypoxanthine and a Cypridina luciferin analog, 2-methyl-6-phenyl-3-7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA). Vitamin E, vitamin C and their related compounds competed with CLA to react with superoxide and reduced the chemiluminescence intensity. From a kinetic analysis of the effect of addition of these compounds on the chemiluminescence intensity, the rate constants for their interactions with superoxide were measured at 25 degrees C and pH 7.8. The rate constants were obtained as 3.3 x 10(5) and 1.7 x 10(4) M-1 s-1 for ascorbate and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, respectively, and also as 4.9 x 10(3) and 4.5 x 10(3) M-1 s-1 for alpha-tocopherol incorporated into soybean and dimyristoyl phosphatidylcholine liposomal membranes, respectively. It has been shown that this method is a sensitive and a quick method which can be applied for measurement of the reactivities of various natural and synthetic compounds toward superoxide. In addition it has been shown that this method can also be applied to the heterogeneous system as well as homogeneous solution, which makes it more versatile and useful for the study in biochemistry.


Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.


Diacylglycerol kinase in plasma membranes from wheat.

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg2+ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl2. The Mg2+ requirement could be substituted only partially by Mn2+ and not at all by Ca2+. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.


Photoaffinity cross-linking of acyl carrier protein to Escherichia coli membranes.

Acyl Carrier Protein (ACP) is a small acidic protein which interacts with the various enzymes implicated in the biosynthesis of fatty acids in E. coli. It also interacts with the inner membrane proteins implicated in the biosynthesis of phospholipids. Samples of radioactive ACP were prepared with high specific activities and bearing photoactivable aryl azide derivatives. Two photoactivable reagents were used: para azido phenacyl bromide (pAPA) which reacts with the SH of the ACP prosthetic group and the N-hydroxysuccinimide ester of 4-azido salicylic acid (NHS-ASA) which reacts with the amino groups of the protein. Various methods were used to demonstrate that ACP could be cross-linked specifically to an inner membrane protein of E. coli, most probably to the glycerol-3-phosphate acyl transferase (GPAT). This covalent link should provide a powerful tool for further analysis of the structure of GPAT and its role in phospholipid biosynthesis. These photoactivable aryl azide derivatives of ACP could also be very useful for studying the interaction of ACP with the soluble enzymes implicated in fatty acid biosynthesis.


Factor IXi-antithrombin (IXiAT) and thrombin-antithrombin (TAT) complexes in lung cancer patients.

Coagulation activation frequently occurs in cancer patients, resulting in thromboembolic complications and/or intravascular coagulation activation. The mechanisms leading to these alterations still are poorly understood. One explanation for the coagulation activation in malignant diseases is the presence of a direct factor X-activating cancer procoagulant. Coagulation activation in lung cancer patients develops at earlier stages than factor X activation; we demonstrated increased factor IXiAT complexes in addition to elevated TAT complexes. The increases of factor IXiAT complexes were not dependent upon the stage of the disease. In contrast, TAT complexes were higher in patients suffering from advanced pulmonary non-small cell carcinoma than in patients with limited disease. In conclusion, coagulation activation in pulmonary cancer patients occurs at earlier steps in the coagulation cascade than factor X activation. While this activation is not dependent upon the stage of the disease, the observation that TAT complexes showed higher elevations in patients with advanced than in those with limited pulmonary non-small cell carcinoma could be an indication of a cancer procoagulant that directly activates factor X.


Heparin-associated thrombocytopenia: successful therapy with the heparinoid Org 10172 in a patient showing cross-reaction to LMW heparins.

A patient suffering from heparin-associated thrombocytopenia (HAT), recurrent arteriothromboses, and acute renal failure after treatment with standard heparin is described. He failed to improve when therapy was continued with low-molecular-weight (LMW) heparin (Fragmin, Kabi Pfrimmer, Erlangen, FRG). By means of the in vitro heparin-induced platelet activation (HIPA) assay it was shown that standard heparin and the LMW heparins Fragmin and Fraxiparin (Sanofi Labaz, Munich, FRG), as well as the enoxaparine Clexane (Nattermann, Cologne, FRG), all induced platelet activation with the patient's serum. In contrast, the LMW heparinoid Org 10172 (Organon, Oss, The Netherlands) did not cause platelet activation. When the patient was subsequently treated by parenteral administration of Org 10172 as anticoagulant over a period of several weeks the number of platelets rapidly increased and the patient almost completely recovered. This case shows that strong in vivo and in vitro cross-reactivity between standard heparin and LMW heparins may occur, but can be avoided by the use of a novel heparinoid, Org 10172. The HIPA assay provides a simple and sensitive laboratory method for the choice of an innocuous heparin or heparinoid for continued parenteral anticoagulation.


Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes.

Parathyroid hormone (PTH) binds specifically to the hypertrophic region of growth plate cartilage [16]. This specific binding suggests a role for this hormone in chondrocyte maturation. Enzymatically isolated chick articular and growth plate chondrocytes grown in monolayer culture were used to assay the direct effects of PTH on chondrocytes. The articular chondrocytes were unresponsive to PTH. The growth plate chondrocytes, however, demonstrated a marked mitogenic response to PTH, with a 39-fold increase of [3H]-thymidine incorporation into DNA. PTH also affected matrix production by the growth plate chondrocytes causing a twofold stimulation of proteoglycan synthesis as determined by the rate of 35SO4 incorporated into matrix macromolecules. Furthermore, PTH depressed collagen synthesis as measured by [3H]-proline incorporation. PTH caused a 12-fold increase in intracellular cAMP in growth plate chondrocytes but no increase in the articular cells. This specificity of PTH for growth plate chondrocytes suggests a possible regulatory role in enchondral ossification.


Two biochemical indices of mouse bone formation are increased, in vivo, in response to calcitonin.

In a series of four studies, adult female Swiss-Webster mice were used to measure the effects of salmon calcitonin on two biochemical indices of local and systematic bone formation: (1) skeletal alkaline phosphatase activity--in serum and in extracts of calvaria and tibiae, and (2) calvarial collagenase-digestible protein synthesis--measured, acutely, in vitro. Subcutaneous calcitonin doses ranged from 50 to 400 mU/mouse/day (0.95-18.1 U/kg/day), and treatment schedules were continuous (daily) for 2-14 days, acute, or intermittent (2 days/week for 6 weeks). The effects of calcitonin on these bone formation indices (skeletal alkaline phosphatase and collagenase-digestible protein synthesis) were biphasic with respect to dose and treatment time, being increased in response to short-term, low-dose treatment, but not long-term, continuous treatment. The effects of long-term intermittent calcitonin treatment were dose-dependent increases in skeletal alkaline phosphatase in calvaria and serum (r = 0.948, P less than 0.02, and r = 0.960, P less than 0.01, respectively).


Cancer in children.

Cancer is a relatively unusual disease entity in children. However, its diagnosis often proves devastating for the families of afflicted youngsters. The types of cancer seen in children are very different from those found in adults. Primarily, they include leukemias, lymphomas and brain tumors. In most cases, the cause(s) of these cancers is unknown. Standard treatment modalities include surgery, radiotherapy and chemotherapy. Treatment is generally provided by a multidisciplinary team which deals with attempts to eradicate cancer and provides support when there are complications of disease and treatment. The team also pays attention to the psychosocial needs of the child and the entire family unit. With recent improvements in cancer therapy, many children are now considered to be "cures." Treatment is tailored to the different subgroups of each disease entity in order to improve response. Research on the etiology of cancer and the treatment of patients with resistant disease is continuously being carried out by cooperative groups. Attention is being directed at the long-term complications occurring in the increasing number of children who have been cured of this still devastating disease.


Peripheral nerve injuries in children with traumatic brain injury.

The relationship between traumatic brain injuries and associated peripheral nerve injuries has not been previously studied in children in the rehabilitation setting. One hundred and fifty-seven consecutive admissions of brain-injured children were included in this prospective study to determine the frequency and pattern of recovery of peripheral nerve injuries in such patients. Twelve patients met the clinical diagnostic criteria for peripheral nerve injury. Electrodiagnostic studies confirmed the clinical diagnosis in 11 (7%) patients. Grading of spontaneous activity and electrodiagnostic study of the contralateral limb were used to differentiate electromyographic abnormalities due to upper motor neuron lesion and nerve injury. Electrodiagnostic study was useful to confirm clinical diagnosis and predict prognosis of nerve injuries. Two patients developed preventable pressure neuropathy. Eight patients showed moderate or full recovery in 9-10 months post-injury. All children with severe brain injuries should be evaluated for concomitant peripheral nerve injuries.


The association between cholangiocarcinoma and hereditary nonpolyposis colorectal carcinoma.

Eighteen patients with a biliopancreatic carcinoma in 15 different cancer family syndrome (CFS) families were studied. Only families with three or more first-degree relatives with colorectal carcinoma were included, and other characteristics of CFS were required in at least two cases. In 14 patients, the histologic specimen was available for reevaluation. In 11 (79%), the tumor was confirmed as a carcinoma of the biliary tract or papilla of Vater. In three (21%), carcinoma of the pancreas was the most probably alternative. In all four patients without histologic reevaluation, the diagnosis had been carcinoma of the biliary tract. This study suggests that carcinoma of the biliary tract or papilla of Vater is associated more commonly with CFS than with carcinoma of the pancreas. In this respect, CFS resembles familial adenomatosis coli, in which this association previously has been established.


Clinical and histopathologic evaluation of the expression of Ha-ras and fes oncogene products in lung cancer.

The expression of Ha-ras and fes oncogenes was investigated with the immunohistochemical method in formalin-fixed, paraffin-embedded tissue specimens of 147 lung carcinomas. Positive immunoperoxidase reactions for Ha-ras p21 were found in 80.5% of the adenocarcinomas, 39.5% of the squamous cell carcinomas, 21.4% of the large cell carcinomas, and 15.4% of the small cell carcinomas; those for fes P85 were found in 51.2% of the adenocarcinomas, 26.3% of the squamous cell carcinomas, 35.7% of the large cell carcinomas, and 15.4% of the small cell carcinomas. Both Ha-ras p21 and fes P85 were expressed most frequently and most strongly in adenocarcinoma. In addition, adenocarcinoma showed significantly higher incidence of concomitant expression of Ha-ras p21 and fes P85 as compared with other histologic types of lung cancer. Thus, the authors suggest that the cooperative effects of Ha-ras and fes oncogenes are especially important in the carcinogenesis of adenocarcinoma. In adenocarcinoma, the incidence and grade of Ha-ras p21 expression increased with the degree of histologic differentiation, suggesting that Ha-ras oncogene might be related to cellular differentiation. Papillary adenocarcinoma showed more frequent Ha-ras p21 expression in comparison with acinar adenocarcinoma. In well- or moderately differentiated adenocarcinoma, the incidence and grade of Ha-ras p21 immunoreactivity in the cases with poor prognosis were significantly higher than in those with good prognosis if other major prognostic factors were equivalent in the two groups. The authors propose that the expression of Ha-ras p21 may be one of the useful prognostic factors in such carcinomas.


Low incidence of human papillomavirus type 16 DNA in bladder tumor detected by the polymerase chain reaction.

Human papillomavirus (HPV) detection was done using the polymerase chain reaction technique on tumor tissue from 44 patients with transitional cell carcinoma of the urinary bladder. Only one of the 44 was associated with HPV infection. The HPV-positive patient was not known to have immunodeficiency or genital warts, and the tumor was not morphologically different from the other tumors. Control experiments excluded the possibility that this finding was caused by contamination of the sample. This study confirms that HPV infection is a rare condition in bladder carcinoma.


A phase II randomized study comparing sequential and combined intraarterial cisplatin and radiation therapy in primary brain tumors. A Southwest Oncology Group study.

The Southwest Oncology Group conducted a trial of intra-arterial cisplatin (150 mg intravenously every 21 days for 2 doses) administered concomitant with or before radiation therapy. Because of technical difficulties cannulating the artery, 27 of 33 eligible patients were able to receive the cisplatin by arterial injection. Five patients died before completion of the study, and 11 patients in each treatment arm were evaluable for response. Three patients (27%) had an objective tumor regression in each treatment arm. Thromboembolic problems complicated 8 of 57 (14%) chemotherapeutic courses. The median survival for the 33 eligible patients was comparable for both groups: 10.8 months in the concomitant treatment arm and 9.6 months with sequential therapy. Problems related to drug administration and toxicity made it impossible to determine the role of intraarterial cisplatin in the initial management of primary high-grade gliomas.


Establishment and characterization of a human T-cell lymphoblastic lymphoma cell line (HT-1) carrying an inversion of chromosome 14.

A new human lymphoblastic lymphoma cell line was established (designated HT-1) from the pleural fluid lymphoma cells of a patient with lymphoblastic lymphoma of T-cell type. The HT-1 cells expressed CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD57, and terminal deoxynucleotidyl transferase (TdT) but lacked B-cell-associated antigens and myeloid-associated antigens. In addition, HT-1 cells had rearranged T-cell receptor (TCR) beta-chain gene and gamma-chain gene but retained germlines of immunoglobulin (Ig) heavy chain gene. These findings indicate that HT-1 cell line represents a common thymocyte in the T-cell lineage. Cytogenetic studies revealed that HT-1 cells carry an inversion (inv) of the long arm of chromosome 14. This cell line is the second T-cell line carrying inv(14) chromosome and may be useful for the molecular investigation of the cytogenetic break points of inv(14).


Paraneoplastic encephalomyelitis. Dramatic response to chemotherapy alone.

Neurologic paraneoplastic syndromes are usually a debilitating and untreatable manifestation of malignancy. The case is presented of a woman with severe paraneoplastic encephalomyelitis that was characterized predominantly by cerebellar degeneration associated with small cell lung cancer, both of which responded rapidly to cytotoxic chemotherapy alone. She is alive with some neurologic residua but no signs of recurrent cancer more than 2 years after diagnosis. Recommendations for aggressive management of this rare but disabling syndrome are outlined.


Lack of modulation effects of praziquantel on DMN-induced lesion development in the Syrian hamster liver.

The effects of repeated praziquantel administration subsequent to dimethylnitrosamine (DMN) treatment of Syrian hamsters were investigated. The antihelminthic drug was given (200 mg/kg body wt. as a suspension in corn oil, by i.g. intubation) 11 times at 2 week intervals starting at week 4 after initial 20 mg/kg DMN i.p. injections at weeks 0 and 2. Sacrifice at week 28 revealed no differences in either hepatocellular or cholangiocellular lesion development between carcinogen-initiated groups with or without antihelminthic treatment. No lesions were observed in the praziquantel alone or untreated groups. The results thus indicate no promotion potential for praziquantel on nitrosamine-induced lesions in the hamster liver.


Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease.

In man, Hodgkin's disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.


The a mating type locus of U. maydis specifies cell signaling components.

The a mating type locus of the phytopathogenic fungus U. maydis controls fusion of haploid cells and filamentous growth of the dikaryotic mycelium. The a locus exists in two alleles, termed a1 and a2, which are defined by nonhomologous DNA regions comprising 4.5 kb for a1 and 8 kb for a2, flanked by identical sequences. Based on functional assays, mutants, and sequencing, we demonstrate that the mating type in each allele is determined by a set of two genes. One encodes a precursor for a lipopeptide mating factor, and the other specifies the receptor for the pheromone secreted by cells of opposite mating type. Thus, U. maydis employs a novel strategy to determine its mating type by providing the primary determinants of cell-cell recognition directly from the mating type locus.


Functional antagonism between c-Jun and MyoD proteins: a direct physical association.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.


Temperature-sensitive mutations in the III-IV cytoplasmic loop region of the skeletal muscle sodium channel gene in paramyotonia congenita.

Paramyotonia congenita (PMC), a dominant disorder featuring cold-induced myotonia (muscle stiffness), has recently been genetically linked to a candidate gene, the skeletal muscle sodium channel gene SCN4A. We have now established that SCN4A is the disease gene in PMC by identifying two different single-base coding sequence alterations in PMC families. Both mutations affect highly conserved residues in the III-IV cytoplasmic loop, a portion of the sodium channel thought to pivot in response to membrane depolarization, thereby blocking and inactivating the channel. Abnormal function of this cytoplasmic loop therefore appears to produce the Na+ current abnormality and the unique temperature-sensitive clinical phenotype in this disorder.


Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.


Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member.

Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have been 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.


TNF-alpha differentially regulates Ia antigen expression and macrophage tumoricidal activity in two murine macrophage cell lines.

We have assessed tumor necrosis factor-alpha (TNF-alpha) production and its autocrine effects on activation in two murine macrophage cell lines which have distinct responses to the activation stimuli interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), and compared these responses to those observed in thioglycollate-elicited peritoneal macrophages. IFN-gamma induced TNF-alpha production in RAW 264.7 cells and this induction was regulated at the transcriptional level. IFN-gamma did not stimulate TNF-alpha production in either WEHI-3 cells or peritoneal macrophages, although MHC class II antigen expression was induced. LPS stimulated TNF-alpha production in the RAW 264.7 cell line and peritoneal macrophages; however, no TNF-alpha was detected in WEHI-3 cells activated with LPS. We also assessed the ability of endogenous TNF-alpha to serve as an autocrine regulator of two aspects of IFN-gamma-mediated macrophage activation, namely, induction of antibody-independent tumoricidal activity and induction of MHC class II antigen expression. These studies revealed that TNF-alpha could act synergistically or antagonistically with IFN-gamma in the regulation of these two functions, depending on both the macrophage population used and the function assessed. The results of our experiments suggest that the mechanism of induction of TNF-alpha production by IFN-gamma or LPS, and the ultimate autocrine contribution of such TNF-alpha to a given activation response, is dependent on the activated macrophage target population under analysis. The WEHI-3 and RAW 264.7 cell lines provide a model system for comparative exploration of the mechanistic basis of this differential regulation.


Cholera toxin promotes the proliferation of anti-mu antibody-prestimulated human B cells.

The predominant effect of cholera toxin (CT) on cell growth has been postulated to be inhibitory as a result of its induction of intracellular cAMP. We have recently reported that CT selectively enhances surface DR expression while it inhibits anti-mu antibody-induced B lymphocyte proliferation. In the present series of experiments we studied the effect of CT on in vitro preactivated highly purified (greater than 95% CD20+) human B cells. Cholera toxin enhanced thymidine incorporation of anti-mu antibody-preactivated but not of Staphylococcus aureus Cowan I or PMA + ionomycin-preactivated B cells. Concentrations of 100 pg/ml CT stimulated an enhancement of thymidine incorporation equivalent to that of optimal doses of BCGF. The growth factor-like effect of CT required the complete molecule, since binding of purified B subunit (B-CT) to GM1 ganglioside by itself did not reproduce the holotoxin effect. Moreover, B-CT pretreatment of anti-mu antibody-primed cells completely neutralized the holotoxin-enhancing effect. Both PGE2, a physiological agent that stimulates intracellular cAMP elevation, and the cAMP analogue, 8-bromo-cAMP, mimicked the growth-promoting effect of CT. However, the ED50 of CT required to augment proliferation in anti-mu antibody-preactivated human B cells was approximately 100 times less than the ED50 for cAMP formation. These results demonstrate a specific growth factor-like promoting effect of CT on sIg-preactivated highly purified human B cells that may be mediated at least in part through elevation in intracellular cAMP levels. Increased DR expression and stimulation of growth of sIg preactivated B cells may explain some of the adjuvant properties of CT following orally or parenterally administered antigens.


The effect of a shift from a mixed diet to a lacto-vegetarian diet on human urinary and fecal mutagenic activity.

This is an investigation of the effects of a shift from a well-balanced mixed diet to a lacto-vegetarian diet on the mutagenic activity in urine and feces. The participants were 20 normal-weight, non-smoking subjects (4 men and 16 women, mean age 44 years, range 27-61 years). The fecal samples were assayed for direct-acting mutagens with the fluctuation test for weak mutagens and the urinary samples were assayed with the same assay but with a metabolic activation system, a so-called S9 fraction. The switch from a mixed diet to a lacto-vegetarian diet was not a shift from a so-called high to a low risk diet for colon cancer but rather from a 'medium high risk diet' to a 'low risk diet', even though there were significant changes in nutrients and food components between the two diets. There was a decrease in fat (P = 0.009) and protein intake (P = 0.04) and an increase in total carbohydrate (P = 0.001), fiber (P = 0.001), calcium (P = 0.006) and vitamin C intake (P = 0.019). Among the food preparation methods the use of frying decreased (P = 0.02) and the habit of eating a new vegetable meal increased (P = 0.05). Three months after the dietary shift the concentration of fecal direct-acting mutagens decreased significantly (P less than 0.05), though the total mutagenic activity excreted in feces per 24 h was not different between the two diet periods. Both the concentration and the total amount of promutagens in the urine were decreased after 3 months on the lacto-vegetarian diet. The decrease in fecal mutagenic activity might be explained by a higher fiber intake, which leads to higher water content in feces and thereby a dilution of fecal mutagenic compounds.


Regulation of gap junctional communication in Syrian hamster embryo cells by retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.

Retinoids enhance the frequency of Syrian hamster embryo (SHE) cell colonies with transformed morphology in a similar way to tumor-promoting phorbol esters. The present study shows that retinoids are also potent inhibitors of gap junctional intercellular communication in SHE cells at noncytotoxic concentrations. This is an apparent contrast to the results observed in transformation systems using the mouse cell lines C3H10T1/2 and BALB/c 3T3, where retinoids have been found to reduce the induction of transformation, and also to enhance gap junctional cell communication. Retinoids are thus potent modulators of transformation and cell communication in three transformation systems. For all three cell types, enhancement of communication by retinoids is related to reduced transformation, and inhibition of communication to enhanced induction of transformation. Communication in the SHE cells is completely blocked following 1 h exposure to 30 microM retinoic acid, while concentrations of 0.3-15 microM results in a gradual down-regulation of communication during 1-5 h exposure. Removal of retinoic acid results in complete restoration of communication to control values within a few hours. Primary SHE cells and the cell line BPNi show similar sensitivity for inhibition of communication after exposure to retinoic acid, while BPNi cells are far more sensitive to inhibition of communication by 12-O-tetradecanoylphorbol-13-acetate (TPA) than primary SHE cells. Retinoic acid does not induce inhibition of epidermal growth factor binding, potentiate adenylate cyclase activation or enhance arachidonic acid release, as does TPA, suggesting different mechanisms of action.


Spin-labeled phorbol esters and their interactions with cellular membranes--IV. Lipophilic binding and molecular orientation of spin-labeled phorbol-12,13-diesters in human erythrocyte membrane.

In human erythrocyte membranes, membrane binding of spin-labeled TPA-analogous phorbol (doxyl)esters [(n,m)PA] was investigated during measurement of the kinetics of the decay of their electron paramagnetic resonance signal by ascorbate reduction. In membrane-bound (n,m)PA the reduction rate was dependent of the position of doxyl in the aliphatic chain of their 12-O-acyl moiety. To describe quantitatively the reaction kinetics observed, two hypotheses (models) were developed and used. Model 1 is based on the assumption that ascorbate reduction takes place in the extracellular space. In this case the experimental data could be fitted by the partition and permeability coefficients of (n,m)PA determining model 1 only, if non-realistic values of these parameters were used. The more refined model 2, corresponding to a bilayer membrane structure, assumes the reduction to take place in the hydrophilic region of the membrane. Assuming a finite probability of finding the doxyl group within the hydrophilic membrane region, model 2 describes quantitatively the dependence of the reduction rate on the position of the doxyl in the aliphatic chain of the (n,m)PA used. From the validity of this model it may be postulated that the molecular orientation of TPA-analogous (n,m)PA in the bilayer membrane is determined by an anchoring of their lipophilic ester moiety in the lipophilic region of the membrane bilayer, thus locating the hydrophilic phorbol moiety within the hydrophilic region of the membrane. With regard to the well-known categories of non-specific versus specific binding of bioactive phorbol esters to protein kinase C/membrane complexes it is deduced that anchoring of (n,m)PA (and hence TPA) in the hydrophobic interior of the membrane structure may be the molecular equivalent of their non-specific binding.


Spin-labeled phorbol esters and their interactions with cellular membranes--V. Electron paramagnetic resonance of spin-labeled phorbol-12,13-diesters bound to their receptors in mouse brain particulate fraction.

The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (approximately 10 nmol per animal; approximately 10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 x 10(-12) mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a decreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.