Evidence for transfer of cellular and humoral immunity to cytomegalovirus from donor to recipient in allogeneic bone marrow transplantation. In order to study the importance of the immune status of the donor in the development of immunity after allogeneic bone marrow transplantation (BMT), we monitored 23 cytomegalovirus (CMV) antibody-positive BMT recipients for humoral and cellular immunity to CMV, of whom 12 had a CMV antibody-positive and 11 a CMV antibody-negative marrow donor. Lymphocyte proliferation to CMV recovered significantly earlier after BMT in recipients of marrow from a CMV+ donor (10.4 weeks after BMT) compared with the recipients of marrow from CMV- donors (16.7 weeks after BMT, P less than 0.05). This seemed to be specific, as lymphocyte proliferation to phytohaemagglutinin and Candida were not different between the to groups. IgM responses after active infection were seen in both groups, but initial IgG rises without IgM were seen only in recipients of marrow from CMV+ donors (P less than 0.05). Lymphocyte proliferative or humoral immune responses to CMV were not detected in any of the patients in a control group consisting of nine CMV- recipients. These results indicate that T cell memory to CMV is transferred with donor marrow from CMV+ donors, leading in most patients to direct IgG anti-CMV responses and to quicker recovery of cellular immunity to CMV.


Alpha-1 anti-trypsin and CD30 expression occur in parallel in activated T cells. We have performed an immunohistochemical study of immunoblastic T cell lymphoma and enteropathy-associated T cell lymphoma for alpha-1 anti-trypsin and CD30. Cytoplasmic staining for alpha 1 anti-trypsin is present in the malignant cells in both types of T cell neoplasm which also express CD30, a marker of lymphoid activation. Peripheral blood T lymphocytes on stimulation with mitogen also show granular cytoplasmic expression of alpha 1 anti-trypsin. Time course studies show that this parallels the expression of CD30. Alpha 1 anti-trypsin expression appears therefore to be associated with activation in T cells. Further studies of sub-fractionated T lymphocytes in vitro suggest that the expression of alpha 1 anti-trypsin on activation is not restricted to an individual lymphocyte subset.


Ranitidine inhibits adrenergic transmission in the rat isolated anococcygeus muscle. 1. The effects of ranitidine on adrenergic transmission in the rat isolated anococcygeus muscle were investigated. 2. Cumulative doses (2-8 mmol/L) or ranitidine produced a concentration-dependent inhibition of motor responses of the rat isolated anococcygeus muscle evoked by field stimulation (20-25 V, 10 Hz for 10 s, 1 ms pulse width) every 2 min, but also potentiated the contractile response to exogenous noradrenaline (5 mumol/L). The inhibited motor responses recovered rapidly and completely after washing out ranitidine. 3. 4-Aminopyridine (100 mumol/L) effectively reversed the partially inhibited (55% or greater) motor responses. 4. The results strongly suggest that ranitidine can inhibit adrenergic transmission in the anococcygeus muscle by a prejunctional mechanism with, presumably, consequent development of supersensitivity of the effector cells to noradrenaline.


Captopril magnifies the increase in angiotensin I-converting enzyme activity in rats with aminonucleoside nephrosis. 1. Serum, tissue and urine angiotensin I-converting enzyme (ACE) activity was estimated in the following groups of rats: saline-injected rats (controls); captopril-treated (CAP) control animals (CONTROL-CAP); puromycin aminonucleoside (PAN)-induced nephrotic syndrome (NS); and CAP-treated animals with NS (NS-CAP). 2. Serum ACE activity increased in the CONTROL-CAP, NS, and NS-CAP groups. The increase in the NS-CAP group was significantly higher compared with the NS or CONTROL-CAP groups. 3. In the CONTROL-CAP group, tissue ACE decreased in brain, heart and adrenal glands, and remained unchanged in the lung, testis, kidney, small intestine and liver. In the NS group, tissue ACE activity increased in the lung and testis, decreased in the brain and heart, and remained unchanged in the small intestine, adrenal glands, kidney and liver. Tissue ACE activity increased significantly in the NS-CAP group compared with the other groups. This increase in tissue ACE may contribute to an increase in the serum ACE activity in the NS-CAP group compared with the NS group. 4. Urine ACE activity increased in the NS and NS-CAP groups, although the rise in the NS-CAP group was significantly higher. The urine ACE correlated significantly with the circulating levels of this enzyme in the NS and NS-CAP groups. The loss of ACE in the urine in the presence of an increased serum ACE activity indicates that the biosynthesis of tissue ACE and its release into the bloodstream must be elevated.


Bronchodilatory activity and pharmacokinetics of new xanthines in guinea-pigs. 1. The in vitro biological activities and the effect of protein binding on the relaxant effects in vivo of N-3-alkylxanthine and N-3-alkyl-N-1-methylxanthine derivative were investigated in guinea-pigs. 2. A significantly positive correlation was observed among the in vitro muscle relaxant activity, the cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE) inhibitory activity and the protein-binding potency of xanthine derivatives. However, there was a weak relationship between these activities and affinity for adenosine receptors. 3. When theophylline, enprofylline and 1-methyl-3-propylxanthine (MPX) were injected intravenously in guinea-pigs, their ED50 values were 6.1, 3.3 and 1.0 mg/kg, respectively. Plasma concentrations of these drugs obtained following the intravenous injection of the ED50 approximated the theoretically effective concentration (EC50) predicted from both the relaxant effects in vitro and the protein binding parameters. A good linear correlation was observed between bodyweight in four species (rats, guinea-pigs, rabbits and humans) and certain pharmacokinetic parameters of enprofylline and theophylline. 4. The present study indicates that differences in the relaxant effects of these drugs in vitro and in vivo can be explained in part by protein binding, and that the protein binding of these xanthine bronchodilators is an important determinant for their pharmacological activity. Guinea-pigs provide a useful model for studying pharmacodynamic-pharmacokinetic relationships of new bronchodilators.


Modelling of frequency-dependent effects of lignocaine homologues on the maximum upstroke velocity of action potentials in guinea-pig papillary muscles. 1. The effects of 14 lignocaine homologues on the maximum upstroke velocity (Vmax) of the action potentials (AP) were studied in guinea-pig papillary muscles. These drugs possess one, two or three methyl groups in different positions: an ortho-chloro, -carbomethoxy or -ethyl group instead of an ortho-methyl group; or an N-butyl group instead of an N-diethyl group in lignocaine molecules. 2. At 50-100 mumol/L, six drugs possessing two ortho substituents (but not the other eight) reduced Vmax more prominently at 2-4 Hz than at 1 Hz, and slowed the time courses of recovery of the premature responses. None of the drugs affected resting potential. 3. Besides the two-state piecewise exponential model (models I and II) frequently used, a time-dependent and time-independent, two-state model (model III) was formulated and applied to these experimental data. The above two groups were effectively distinguished by the difference of the estimated association and dissociation rate constants (model II) and equilibrium constants for phasic state (model III) and for resting (model II) or tonic (model III) states. 4. The equilibrium constants for resting or tonic state correlated well with log P (where P = the n-octanol: water partition coefficients), but correlated better with an indicator variable that denotes the existence of two ortho substituents, suggesting the importance of the contribution of steric factors to the activity.


Benign vulvar tumors.

Benign vulvar tumors. Benign tumors and "dark lesions" accounted for 22% of vulvar disease seen in the Vulvar Clinic at the Milwaukee County Medical Complex over an 8-year period. Biopsy confirmation was obtained for 269 lesions. The order of frequency of lesions in this study was as follows: epidermal inclusion cyst, lentigo, Bartholin's duct obstruction, carcinoma in situ, melanocytic nevi, acrochordon, mucous cyst, hemangiomas, postinflammatory hyperpigmentation, seborrheic keratoses, varicosities, hidradenomas, verruca, basal cell carcinoma, and, last, unusual tumors such as neurofibromas, ectopic tissue, syringomas, and abscesses. The variability in clinical appearance of vulvar tumors suggests that biopsy confirmation should be obtained on all lesions for which there is the least doubt regarding the diagnosis.


Vulvar manifestations of human papillomavirus infection. As technology continues to improve, we are increasingly able to detect the presence of HPV in both clinically diseased and histologically and clinically normal vulvar tissue. Unfortunately, the technology for effective treatment of these conditions has not advanced as rapidly. Still, fundamental information about the interaction of HPV with the host cell continues to be collected and will provide us with an increased understanding of the importance of latent infection and its progression to clinical vulvar disease. There are a host of therapeutic modalities available for the treatment of HPV-associated conditions of the vulva, ranging from simple topical medication like podophyllin to sophisticated combination therapies involving carbon dioxide laser destruction in conjunction with interferon. Regrettably, there is no "magic bullet" that is totally effective for any of the HPV-associated vulvar diseases. This makes it more important to weigh the benefits and morbidity of treatment regimens before they are used. Until the success rate of available therapies improves, it is important to have definitive goals for therapy, because it is currently impossible to completely eradicate HPV from tissues. Examples of therapeutic goals might range from removal of visible lesions or treatment of symptomatic areas to treatment of premalignant or malignant lesions. At present, perhaps the most important service we can provide for patients is close follow-up of HPV-associated vulvar diseases in an attempt to prevent the development of invasive malignancy. Regardless of the choice of treatment, the primary objective of the clinician should be to help the patient but to do no harm.


Ambulatory strain-gauge plethysmography and blood volume scintimetry for quantitative assessment of venous insufficiency. In the present study, a strain-guage plethysmographic method (ASGP) is compared to an isotope plethysmographic method (ABVS), in order to evaluate how an externally recorded volume change corresponds to blood displacement within the leg during exercise. The coefficient of correlation for RT (venous return time) was 0.67 (P less than 0.001) and for EV (expelled volume) it was 0.61 (P less than 0.001). The RT values were similar by the two methods both in healthy controls and in patients with Chronic Venous Insufficiency (CVI). The corresponding EV values showed the same pattern in controls and patients but the values differed substantially. By ASGP the value was approximately 2 ml per 100 ml tissue compared to 33 ml per 100 ml blood by the ABVS method. Both methods could clearly distinguish normal controls from patients with chronic deep venous insufficiency, but ABVS could only make this distinction when the ankle area of measurement was applied.


Primary cerebral anaplastic T-cell-lymphoma (type Ki-1): review and case report. We describe the clinical course of a 20-year-old man who suffered generalized convulsive seizures with postictal aphasia and hemiparesis of the right side. Computed tomography (CT) displayed a left postcentral lesion with prominent perifocal edema and only a little contrast medium enhancement. The completely removed tumor proved to be a primary cerebral non-Hodgkin lymphoma consisting of T-cells. Only ten days after the operation the patient once more presented a clinical deterioration. A nuclear magnetic resonance imaging (MRI) displayed an annular structure in the area previously operated upon, suspected to be an abscess. The second operation disclosed a large recurrence of the primary T-cell lymphoma extending diffusely into the white matter. On account of the rapid recurrence, a whole brain irradiation was started twelve days after the second operation. Four cycles of chemotherapy followed. Immunohistochemical studies of the anaplastic large lymphoma cells showed staining with the pan T-cell markers (UCHL1, CD3) and with the CD30 (Ki-1) antibody. The B-cell markers (L26, LN1) were negative. The EMA (epithelial membrane antigen) was only partially expressed. Further investigation excluded the presence of systemic lymphoma manifestation. 24 months after the last operation the patient remained free of symptoms. The last MRI displayed no evidence for the recurrence of a lymphoma. In reference to this unusual clinical course the few previously reported cases of the extremely rare primary cerebral T-cell lymphoma are reviewed.


Recommendations: HIV/HBV infected health care workers. Connecticut Department of Health Services. These recommendations have been developed in response to a congressional mandate and to public concern about transmission of HIV from health care workers to patients. Hepatitis B is also addressed in the recommendations. Both viruses are transmitted through blood-to-blood contact. The likelihood of transmission of HBV is very low, for HIV extraordinarily remote. The following are action steps to reduce this risk further: 1. The commissioner charges the professional associations and institutions to develop plans to educate their members, nonmembers, and employees on universal precautions, procedural modifications, and hepatitis B vaccination. They accept the charge. 2. The associations and institutions are charged with monitoring and assuring the compliance of their members, nonmembers and employees with the precautions. The Department of Health Services health facility licensing programs will incorporate review of infection control precautions into site reviews. Complaints of an institution's or individual practitioner's failure to follow the precautions will be investigated by the Department of Health Services Division of Hospital and Medical Care or Division of Medical Quality Assurance, respectively. 3. The professional associations will submit training plans and progress-to-date to the Commissioner of the Department of Health Services by 30 June 1992 and reports of training and assurance activities on 1 January 1993 and annually thereafter. 4. The department will not list specific hazardous procedures but will judge each case on an individual basis. 5. The department will not require health care workers to undergo HIV or HBV testing. It is recommended that workers who are at risk for HIV or HBV infection because of occupational exposure or personal behaviors be tested voluntarily. 6. The department recommends that infected workers seek advice from a state appointed and authorized review panel. The panel will review the practices of the worker, advise on infection control practices and monitor to assure compliance. Infected workers will be advised regarding notification of patients on a case-by-case basis. Institutional or professional association-based panels can also be consulted.(ABSTRACT TRUNCATED AT 250 WORDS)


Involvement of autophagic degradation in ACTH-induced skeletal muscle atrophy. Intracellular autophagic breakdown was investigated in skeletal myocytes undergoing atrophy under the influence of ACTH. Adult male Sprague-Dawley rats received s.c. injections of ACTH (12.5 U/kg body wt) or physiological saline as control twice daily. 5 experimental and 5 control animals were sacrificed by perfusion fixation after 6, 9, and 12 days of treatment. While the weight of the left pectoralis minor muscle decreased significantly in the ACTH-treated animals compared with controls, the volume fraction of autophagic vacuoles, as determined by quantitative electron microscopy, increased significantly after 9 days (2.6-fold) and 12 days (2.9-fold) in the atrophying skeletal myocytes of experimental animals compared with controls. Only non-myofibrillar components were detected as contents of autophagic vacuoles. Since, however, the ratio of myofibrils to other cytoplasmic components remained constant, the degradation of myofibrils seems to occur via a non-lysosomal pathway. The data suggest that the breakdown of cytoplasmic components by cellular autophagy is important for the development of skeletal muscle atrophy as observed following long-term ACTH treatment or in Cushing's syndrome.


Differential effects of butylated hydroxytoluene analogs on bull sperm subjected to cold-induced membrane stress. Previous reports established that butylated hydroxy toluene (BHT) minimized cold-induced membrane rupture in sperm from several species. No data regarding the specificity of its effect is available. In this study 25 BHT analogs were tested for their effect on bovine sperm membrane stability. Fourteen were membrane lytic at 25 degrees C and 6 were neither membrane lytic nor membrane stabilizing. The remaining 5 compounds, a family of 2,6-tert-butyl phenols with substitutions at position 4 of hydrogen, methyl (BHT), ethyl, butyl, hexyl, or octyl, afforded effective membrane protection to cold shock. Since membrane protection is a function of both the ability of a compound to partition into the membrane and a molecule's effectiveness once there, an analysis of each analog's membrane partitioning, assessed by measuring the cellular analog/cholesterol ratio, showed the following extents of transfer for the analogs: ethyl = butyl greater than methyl = hydrogen greater than hexyl greater than octyl. Thus, an optimum chain length exists for partitioning from micellar donors into cells. A separate experiment established that all analogs, when incorporated in equivalent amounts, protect equally plasma and mitochondrial membranes from cold shock. No effect on acrosomal membrane stability was noted. BHT, but not the other analogs, reduced sperm motility. Addition of egg yolk to extender containing BHT analog protected sperm motility from cold shock but had little effect on membrane stabilization. Analysis of sperm membrane compartments revealed that little to no analog was partitioned into the outer acrosomal membrane or the plasma membrane overlying the acrosome, but rather was localized in other portions of the sperm. We conclude that (a) the effective BHT analogs, if partitioned into the membrane, are indistinguishable with regard to their capacity to eliminate cold-induced membrane lysis; (b) membrane-linked events (e.g., motility) are uniquely disrupted by a subset of this analog family; and (c) when concentrations of egg yolk and BHT analogs are carefully controlled, unique synergistic effects are noted.


Immune-mediated neuropathy and myopathy in post-streptococcal disease: electron-microscopical, morphometrical and immunohistochemical studies. A 22-year-old man suffered from a complete flaccid tetraparesis and an immune complex-mediated rapid progressive glomerulonephritis after group A streptococcal infection. Serum creatine kinase was excessively elevated and myoglobinuria occurred. Nerve conduction studies revealed evidence of axonal neuropathy. Recovery was satisfactory within 18 months. Sural nerve and peroneus muscle biopsies were performed in the 4th and 14th week of the disease. Light microscopy of the sural nerve showed an incipient axonal type of neuropathy in the first biopsy. Ultrastructurally, Wallerian degeneration and endoneurial inflammatory cells were present. In the muscle biopsy, few atrophic fibers and altered blood vessels without further anomalies were found. In the second sural nerve biopsy, macrophages were numerous, some of which were immunoreactive for HLA-DR, and only a few myelinated and some unmyelinated nerve fibers remained. Muscle fibers in the second biopsy showed high-grade atrophy and myofibrillar abnormalities. Immunohistochemistry revealed diffuse endoneurial immunoglobulin deposition in the first sample, while in the later biopsy specimen, deposits of IgG, and kappa and lambda light chains were visible in circumscribed endoneurial areas. Immune-mediated neuropathy and myopathy are not well-known complications of streptococcal disease. This is, to our knowledge, the first detailed report on morphological findings in muscle and nerve in such a disorder.


Induction of stress proteins in SV-40 transformed human RPE-derived cells by organic oxidants. The expression and induction of heat shock proteins (HSPs) were examined in cultured SV-40-transformed human retinal pigment epithelial (RPE)-derived cells following exposure to chemical oxidants. Concentrations of hydrogen peroxide and the organic oxidants tert-butyl hydroperoxide, cumene hydroperoxide and linoleic acid hydroperoxide were used under conditions where cell viability was between 75% and 90% as determined by the trypan blue exclusion tests. The types of HSPs that are either induced and/or elevated from constitutive levels in cultured transformed cells were separated both by SDS-PAGE and by two-dimensional gel electrophoresis. Subsequent immunoblotting was performed with both a monoclonal antibody (MAb C92) specific for only the stress-inducible form of HSP 70, namely HSP 72, and with a monoclonal antibody (MAb N27) specific for both the stress-inducible HSP 72 and its constitutive form, HSP 73. As positive controls for comparison, other types of stressing agents were used that included heat shock at 41 degrees C for various times and exposure to the proline analogue, azetidine-2-carboxylic acid (AzC). Protein immunoblotting analysis demonstrate that: (a) Stress-inducible HSP 72 is present at low levels in non-stressed cultured transformed RPE-derived cells and in fresh bovine retina and RPE, but is not detectable in non-stressed cultured lung fibroblasts until induction with heat shock; and (b) Stress-inducible HSP 72 is elevated from constitutive levels in RPE-derived cultured cells after exposure to various oxidants. After cellular exposure to both organic oxidants and to AzC in the presence of L-[35-S]-methionine, two-dimensional gel electrophoresis confirmed the elevation of newly synthesized HSP 72. Thus, these results indicate that cultured human SV-40-transformed RPE-derived cells are capable of elevated HSP 72 biosynthesis under conditions of oxidative stress produced by organic oxidants.


Na,K-ATPase and phospholipid degradation in bovine and human lenses. Na,K-ATPase, an enzyme intrinsic to the membrane of most cells, is inhibited in cataract. Na,K-ATPase, activity in clear non-cataractous lenses is found predominantly in the lens epithelium. The lens fiber cells would appear to be unique, among mammalian cells in that Na,K-ATPase activity is low if not absent. The study presented here indicates that Na,K-ATPase is present, often in high concentration, but progressively more functionally compromised as the fiber cells mature. The membrane lipid environment as causative agent in the loss of normal function of Na,K-ATPase, is considered in this study. The data indicate a correlation between increasing cholesterol/phospholipid ratio, increasing phospholipase A2 activity and decreasing Na,K-ATPase activity in normal clear lenses. The phospholipase A2 activity is higher in cortex and nucleus than in the epithelium of normal bovine and human lenses. The phospholipase A2 is Ca2+ dependent and may be membrane associated.


[Closure of upper median sternotomy with resorbable polyglycolic acid sutures]. Since 15 years we use absorbable polyglycolic acid sutures (Dexon) for closure of partial median sternotomies. In a consecutive series of 121 refixations, minor superficial wound infections developed in 4 patients. All infections healed completely by local treatment. No major sternal complication occurred. Of 92 patients, who were still living, 82 patients were controlled between 3 and 180 months postoperatively. 80 (98%) sternotomies were consolidated completely; 71 (87%) without any dislocation and 9 (11%) with a small dislocation of the fragments. In 2 cases (2%) an isolated pseudarthrosis of the transverse osteotomy occurred; in both cases the median osteotomy was consolidated completely. In the literature no comparable publication about closure of partial median sternotomies could be found. According to our experiences we consider, that the closure of partial upper sternotomies with polyglycolic acid sutures is as a practical and reliable method.


Isolated or phagocytosed miniature dyskeratotic cells in papilloma virus infection associated with cervical neoplasia. Dyskeratotic cells that occur in squamous cervical epithelium under certain abnormal conditions appear in cervical smears in sheets or aggregates, or as miniature cells, either isolated or phagocytosed. In order to determine the tissue correlates of such cells, 116 cervical biopsies with a histopathologic diagnosis of papillomavirus (HPV) infection were reviewed and compared to previous findings in cytological smears; the patients were 18-65 yr of age. Eighty of the 116 cases had, in addition, cervical intraepithelial neoplasia (CIN). A much higher number of miniature dyskeratotic cells was recorded in the biopsies from patients with both HPV and CIN types of lesions (76.3% of the biopsies had isolated miniature cells and 41.3% showed phagocytosed miniature cells), compared to those with only HPV infection (19.4% with isolated and 5.5% with phagocytosed miniature cells). In addition, in the HPV plus CIN patients, the more advanced the neoplastic lesion, the higher the percentage of cases in which both types of dyskeratocytes are observed. These highly keratinized isolated miniature cells are found in, and are considered to arise from, the deeper layers of the epithelium; when phagocytosed they appear in the center of a concentric arrangement of cells. These cells constitute a pathological entity that can be distinguished from the sheets and aggregates of dyskeratotic cells that come from the superficial layers of parakeratotic epithelia.


Fine-needle aspiration biopsy of Ki-1-positive anaplastic large-cell lymphoma. Five cases of Ki-1-positive anaplastic large-cell lymphoma diagnosed by fine-needle aspiration biopsy are reviewed, and cytologic, histologic, and ultrastructural findings in these cases are correlated. In all cases, the diagnosis of anaplastic large-cell lymphoma was suggested on the basis of the morphological appearance in aspiration smears. This diagnosis was confirmed by immunohistochemistry, which revealed strong positivity of most of the cells by Ki-1 antibody. Two of the lymphomas were T-cell type, one was B-cell type, and the remaining 2 were composed of null cells. In 2 cases, intracytoplasmic inclusions were seen in some of the tumor cells in aspiration smears. These were ultrastructurally correlated with large lysosomal bodies of variable morphology. Fine-needle aspiration combined with immunohistochemistry may be an effective technique for diagnosing this neoplasm.


Asteroid bodies and calcium oxalate crystals: two infrequent findings in fine-needle aspirates of parotid sarcoidosis. We have studied 3 cases of sarcoidosis involving the parotid gland by means of fine-needle aspiration cytology (FNAC). The main findings were noncaseating granulomas, multinucleated giant cells (MGCs), and lymphocytes. In one case MGCs contained asteroid bodies and in another case we observed calcium oxalate crystals (COCs) over both stromal fragments and MGCs. Although nonpathognomonic for sarcoidosis, these 2 findings may help in the diagnosis of this condition. However, both are easily overlooked and must be borne in mind when viewing noncaseating granulomas. Sarcoid granulomas displaying COC must be differentiated from foreign-body granulomas. The aforementioned cytological findings must be assessed in conjunction with clinical findings. Nevertheless, in most cases the diagnosis of sarcoidosis is made by exclusion.


Electron microscopy of fine-needle aspiration biopsy from extragonadal germ cell tumors. We describe five cases of extragonadal germ cell tumor (EGCT) diagnosed by the electron microscope (EM) on cytological material. The clinical diagnosis was incorrect in all cases and EGCT was suspected in two cases; cytological diagnosis by light microscopy confirmed the presence of malignant tumor cells, but did not identify the cytotype/s correctly except in one case. Ultrasonography, laparoscopy, and autopsy (in case 3) excluded a primitive germ cell tumor (GCT). Histology confirmed the EM diagnosis in all cases. EM, even of scanty or necrotic cytological material, is particularly useful for mediastinal and retroperitoneal masses. In case of EGCT, EM can identify the different cytotypes and the different ultrastructural subcellular cytotypes and demonstrates a close relation between seminomatous and nonseminomatous GCT, which could influence their classification and prognosis.


The estimation of mean nuclear volume in the diagnosis of breast carcinoma. Sixty-two cases of breast pathology were randomly selected from the files of the Dunedin Public Hospital for evaluation of mean epithelial nuclear volume. The cases were comprised of both benign and malignant ductal epithelial disease, diagnosed in cytological smears or in histological sections. Nuclear volume in histological preparations was estimated by the stereological technique of point-line intercept measurements to derive volume-weighted mean volumes (vV). An index of the nuclear volume (Vi) of cytology smears was calculated from measurements of nuclear areas by either image analysis Vi(e) or point-line intercepts Vi(p). By all methods of analysis a clear distinction of nuclear volume was found between the benign [means for the cytology were 148 microns 3 (Vi(e)) or 246 microns 3 (Vi(p)), and 203 microns 3 (Vv) for the histology specimens] and malignant diseased cases [means for cytology: 524 microns 3 (Vi(e)) or 886 microns 3 (Vi(p)), and 587 microns 3 (vV) for the histology specimens].


Effect of glucose on Na, K-ATPase activity in cultured bovine aortic endothelial cells. The effect of high concentrations of glucose on Na, K-ATPase activity and the polyol pathway was studied using cultured bovine aortic endothelial cells. Na, K-ATPase activity was expressed as ouabain-sensitive K+ uptake. A significant decrease in Na, K-ATPase activity with an intracellular accumulation of sorbitol was found in confluent endothelial cells incubated with 400 mg/dl glucose for 96 h. However, there was no significant change in the Na, K-ATPase activity or sorbitol content of the cells incubated with 100 mg/dl glucose plus 300 mg/dl mannitol. The decrease in Na, K-ATPase induced by the high glucose concentration was restored by the simultaneous addition of 10(-4) M ponalrestat (ICI 128,436; Statil), an aldose reductase inhibitor. The addition of this agent also significantly reduced the increase in sorbitol induced by high glucose levels. These results suggest that the decrease in Na, K-ATPase activity induced in cultured aortic endothelial cells by high concentrations of glucose may be caused in part by the accumulation of sorbitol.


Effects of WEB 2086, an antagonist to the receptor for platelet-activating factor (PAF), on PAF-induced responses in the horse. Platelet-activating factor (PAF) causes oedema and neutrophil accumulation when injected into the skin of normal horses. PAF is also known to induce aggregation of horse platelets in vitro. The selective PAF receptor antagonist WEB 2086 has now been used to determine whether these effects are mediated by PAF receptor activation. Addition of WEB 2086 to equine platelets in vitro inhibited PAF-induced aggregation in a competitive reversible manner (pA2 = 7.14). Inhibition of in vivo inflammatory responses to PAF occurred after local administration of WEB 2086: wheal formation induced by 0.1 micrograms PAF/site was reduced by 1-10 micrograms WEB 2086/site. PAF (1 micrograms/site)-induced neutrophil accumulation was also inhibited by co-administration of 10 micrograms WEB 2086/site. Systemic administration of WEB 2086 (3 mg/kg iv) to 4 normal ponies inhibited PAF-induced wheal formation and ex vivo platelet aggregation. At 30 min after drug administration the concentration of PAF required to produce a half maximal aggregation response was increased 496 +/- 137 fold. At 6 h the degree of inhibition was markedly reduced and responses had returned to pre-treatment values by 24 h. PAF-induced increases in cutaneous vascular permeability were also reduced by 80% as early as 30 min after iv administration of WEB 2086 in these animals, the inhibition persisting for at least 6 h. These results suggest that in vitro and in vivo responses to PAF in the horse are mediated via activation to PAF receptors. WEB 2086 will therefore be a useful agent for studying the role of PAF in the pathogenesis of equine inflammatory conditions.


Linear and cyclic peptide analogues of the polypeptide cardiac stimulant, anthopleurin-A. 1H-NMR and biological activity studies. A loop corresponding to residues 8-17 in the polypeptide cardiac stimulant anthopleurin-A is known to be important for the cardiostimulant activity of this molecule. To investigate the activity and possible conformations of this loop in isolation, two synthetic peptides have been studied. The first corresponds to residues 6-20 of anthopleurin-A with Cys6 replaced by Thr, and the second to residues 6-21 of anthopleurin-A, with Thr21 replaced by Cys. The introduction of an additional cysteine in the latter peptide enabled an intramolecular disulfide to be formed between the N- and C-terminal residues. Both linear peptides and the disulfide-containing analogue lack the cardiostimulant and Na(+-)-channel binding activity in the parent molecule, anthopleurin-A, indicating that although the loop is important for the function of anthopleurin-A, other regions of the molecule must also be involved in activity. Assignments of the 1H-NMR spectra of both peptides are presented, and their pH and temperature dependences investigated. The results show that the amide protons of Gly5 and Asn11 (corresponding to Gly10 and Asn16 in anthopleurin-A) sample hydrogen-bonded conformations in solution. Based on these NMR data, two regions of non-random structure, encompassing residues 2-5 and 8-11, respectively, are proposed, and the possible involvement of such structures in the activity of anthopleurin-A is discussed.


Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.


Multi-frequency EPR and high-resolution Mössbauer spectroscopy of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). Characterization of a supercluster and superspin model protein. The putative [6Fe-6S] prismane cluster in the 6-Fe/S-containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in 57Fe, and has been characterized in detail by S-, X-, P- and Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution 57Fe Mössbauer spectroscopy. In EPR-monitored redox-equilibrium titrations, the cluster is found to be capable of three one-electron transitions with midpoint potentials at pH 7.5 of +285, +5 and -165 mV. As the fully reduced protein is assumed to carry the [6Fe-6S]3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments: [6Fe-6S]3+, [6Fe-6S]4+, [6Fe-6S]5+, [6Fe-6S]6+. The fully oxidized 6+ state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electron-reduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is low-spin S = 1/2; the remainder has an unusually high spin S = 9/2. The S = 1/2 EPR spectrum is significantly broadened by ligand (2.3 mT) and 57Fe (3.0 mT) hyperfine interaction, consistent with a delocalization of the unpaired electron over 6Fe and indicative of at least some nitrogen ligation. At 35 GHz, the g tensor is determined as 1.971, 1.951 and 1.898. EPR signals from the S = 9/2 multiplet have their maximal amplitude at a temperature of 12 K due to the axial zero-field splitting being negative, D approximately -0.86 cm-1. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of [E/D] = 0.061. A second component has g = 9.7, 8.1 and 6.65 and [E/D] = 0.108. When the protein is reduced to the 4+ intermediate state, the cluster is silent in normal-mode EPR. An asymmetric feature with effective g approximately 16 is observed in parallel-mode EPR from an integer spin system with, presumably, S = 4. The fully reduced 3+ state consists of a mixture of two S = 1/2 ground state. The g tensor of the major component is 2.010, 1.825 and 1.32; the minor component has g = 1.941 and 1.79, with the third value undetermined. The sharp line at g = 2.010 exhibits significant convoluted hyperfine broadening from ligands (2.1 mT) and from 57Fe (4.6 mT). Zero-field high-temperature Mössbauer spectra of the protein, isolated in the 5+ state, quantitatively account for the 0.8 fractional enrichment in 57Fe, as determined with inductively coupled plasma mass spectrometry.(ABSTRACT TRUNCATED AT 400 WORDS)


13C and proton NMR studies of horse cytochrome c. Systematic assignment of methyl and methine resonances in both oxidation states. The CHn groups in the aliphatic side chains of horse cytochrome c have been characterized according to the chemical shifts of both 13C-NMR and 1H-NMR signals, their temperature dependence and the number of attached protons, n. The primary assignments of resonances from the 55 side-chain methyl and the 27 methine groups were obtained directly for the oxidised and the reduced forms. Specific assignments of the 13C resonances were obtained through shift-correlation experiments and comparison with earlier 1H-NMR studies, by further measurements of proton-proton interactions, or by elimination. Comparison of the paramagnetic shifts of carbon and protons indicates a small redox-related change of conformation in the vicinity of Trp59 and a significant expansion of the protein over 30-50 degrees C.


The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones. Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone-induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membrane/mass protein and a change in the intrinsic permeability of the phospholipid bilayer.


Bundling of microtubules by synapsin 1. Characterization of bundling and interaction of distinct sites in synapsin 1 head and tail domains with different sites in tubulin. Synapsin 1 is a nerve terminal phosphoprotein whose role seems to encompass the linking of small synaptic vesicles to the cytoskeleton. Synapsin 1 can join small synaptic vesicles to neuronal spectrin, microfilaments and microtubules; it can also bundle microtubules and microfilaments. In this paper, the mode of interaction between synapsin 1 and microtubules has been investigated. Bundling is shown to be highly cooperative: the apparent Hill coefficient is 3.06 +/- 0.3, and bundling is half-maximal at 0.63 +/- 0.02 microM. Bundling occurs either when whole synapsin 1 preparations (containing monomers and oligomers) or when monomeric synapsin 1 is added to microtubules. However, it is not clear that synapsin 1 remains monomeric in the presence of microtubules. Synapsin 1-microtubule mixtures contain two types of filament. One type is characterised by microtubules often with synapsin 1 bound to their surface. The other type is composed of filaments of diameter 15 +/- 5 nm. This filament type is granular and made up in part of 14-nm-diameter particles. These dimensions are consistent with their being made up of polymerised synapsin 1. It is possible that microtubules induce the polymerisation of synapsin 1. Synapsin 1 had independent tubulin binding sites in the N-terminal head domain and in the C-terminal tail domain. Whole synapsin 1 can interact with tubulin after it has been digested to remove the tubulin C terminus (des-C-terminal tubulin). The interaction of des-C-terminal tubulin with synapsin 1 appears to be via the head domain, since 125I-des-C-terminal tubulin only shows specific binding to the head domain on gel blots. By contrast intact tubulin binds to both head and tail domains. Binding to the tail domain can be inhibited by a synthetic peptide representing the microtubule-associated protein 2 (MAP2) binding site of class II beta tubulin. These results suggest a model for microtubule bundling by synapsin 1 in which independent sites in the head and tail domains of synapsin 1 cross-link microtubules by interactions with two distinct sites in tubulin.


Halothane metabolism. Impairment of hepatic omega-oxidation of leukotrienes in vivo and in vitro. Omega-oxidation of leukotrienes is the initial step of hepatic degradation and thus inactivation of these proinflammatory mediators. Omega-oxidation is followed by beta-oxidation of leukotrienes from the omega-end. After exposure of rats to a single dose of the anesthetic agent halothane, a transient decrease in leukotriene omega-oxidation was induced both in vivo and in vitro. In untreated rats, 44.1 +/- 6.0% of N-[3H]acetylleukotriene E4 injected intravenously was recovered unchanged in bile collected for 60 min in vivo; 46.5 +/- 3.0% was recovered as omega-/beta-oxidation products, of which 24.7 +/- 4.5% were associated with beta-oxidation products only (mean +/- SEM; n = 5). In rats receiving a single dose of halothane 18 h before the experiment, recovery of unchanged N-[3H]acetylleukotriene E4 was significantly increased to 79.8 +/- 4.8%, while the fraction of omega-/beta-oxidation products decreased to 9.0 +/- 1.7% (n = 5); 90 h after exposure to halothane, N-[3H]acetylleukotriene E4 recovery decreased to 30.0 +/- 3.0% and omega-/beta-oxidation products amounted to 49.1 +/- 3.8%; the fraction of beta-oxidation products was significantly increased to 43.1 +/- 3.4% (n = 5). Ten days after exposure of rats to halothane, the recoveries of N-[3H]acetylleukotriene E4, of omega-/beta-oxidation products, and of beta-oxidation products alone, returned to almost normal values. Microsomal fractions obtained from rat hepatocytes catalyzed the NADPH- and O2-dependent leukotriene omega-oxidation in vitro. The formation of omega-hydroxy-metabolites of leukotriene B4, leukotriene E4, and N-acetylleukotriene E4 was decreased by 50% in microsomal fractions obtained from rats 18 h and 90 h after halothane treatment, and returned back to control levels in microsomal fractions obtained 10 days after halothane treatment. The Km value of leukotriene B4 omega-oxidation revealed no significant change in enzyme affinity towards leukotriene B4; in contrast, as reflected by the reduction of the Vmax value by 65%, a decrease in the amount of the active enzyme in microsomes obtained from rats 18 h after halothane treatment was observed. Halothane-metabolism-dependent trifluoroacetylation of hepatic proteins may mediate this process. Thus, the time course of the density on immunoblots of trifluoroacetylated protein adducts paralleled that of the transient decrease in leukotriene omega-oxidation. In contrast to its omega-oxidation, leukotriene B4 synthesis from 5-hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from rats pretreated with halothane. The data suggest that metabolism of halothane causes a transient derangement of hepatic leukotriene homeostasis in vivo.


Changes in membrane fluidity and Na+/K(+)-ATPase activity during human trophoblast cell culture. The human placenta plays an essential role in embryo development, in particular regulating the transport of ions, nutrients and immunoglobulins from the maternal to the fetal circulation. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and elucidated by in vitro studies. The structural stages by which the syncytial trophoblast is formed are not yet understood. In order to clarify the mechanism of trophoblast development, we studied the morphological characteristics of the syncytial trophoblast formation in culture and the functional changes (transport properties and membrane microviscosity) accompanying the structural modifications. By using both 5-nitroxystearate and 16-nitroxystearate as spin labels, we observed an initial increase in membrane order over 0-24 h of culture, which can be associated with two events: recovery of cell membranes from trypsin and initial aggregation of cytotrophoblasts. The similar behaviour of the order parameters determined with both probes indicates that membrane order changes both inside and in the outer part of the lipid bilayer. The subsequent decrease in membrane order observed at 36-48 h might be related to the process of cellular fusion. The increase in sodium/potassium pump activity in the first 24 h of culture might be an expression of cell recovery following trypsin treatment. The subsequent decrease might represent an adaptive mechanism by which metabolic energy is mainly used for morphogenetic changes.


Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa. We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.


Protein-tyrosine phosphorylations induced by concanavalin A and N-formyl-methionyl-leucyl-phenylalanine in human neutrophils. The ability of the lectin concanavalin A (ConA) and N-formyl-methionyl-leucyl-phenylalanine (fMLF) to induce protein-tyrosine phosphorylation in human neutrophils was examined by immunoblot analysis. ConA caused an increase in tyrosine phosphorylation of protein bands with apparent molecular masses of 120, 80, 76, 66 and 40 kDa; on the other hand, fMLF caused an increase in those of only 80-kDa and 40-kDa proteins. These protein-tyrosine phosphorylations were time- and dose-dependent. The tyrosine phosphorylation of 40-kDa protein induced by fMLF was suppressed but that by ConA was not suppressed by pertussis toxin pretreatment. At the same time, pertussis toxin pretreatment also inhibited lysozyme release and aggregation of neutrophils induced by fMLF but did not inhibit those responses induced by ConA. These results suggest that the tyrosine phosphorylation of 40-kDa protein may be involved in a part of neutrophil activation and be regulated via pleiotropic signal transduction pathways. In addition, immunoblot analysis employing antibodies against microtubule-associated protein 2 (MAP2) kinase suggested that this tyrosine-phosphorylated 40-kDa protein might be the MAP2 kinase.


Regulation of gene expression by cytokines and virus in human cells lacking the type-I interferon locus. A number of genes that are induced by type-I interferons are also activated by one or more other inducers, including double-stranded RNA, viruses, interferon-gamma, interleukin-1 and tumor necrosis factor. However, these inducers can also activate the expression of type-I interferons. Thus, the activation of type-I interferon-inducible genes by these other inducers could be direct, or a secondary consequence of the induction of interferon. To distinguish between these possibilities, we have used cell lines lacking all type-I interferon genes to study the direct effect of potential inducers on the expression of 14 interferon-inducible human genes. We show that double-stranded RNA, virus, interferon-gamma or tumor necrosis factor-alpha can act directly to induce specific subsets of type-I interferon-inducible genes in the absence of any possible type-I interferon involvement. The cis-acting element which confers inducibility by type-I interferon has been shown in some cases to confer inducibility by interferon-gamma, double-stranded RNA or virus as well. However, not all promoters containing such an element respond to both interferon and other inducers. Thus, the ability of a given gene to respond to different inducers most likely depends on the exact nature and specific combination of cis-acting elements present in its promoter.


A novel cyclic analog of neuropeptide Y specific for the Y2 receptor. The low-molecular-mass, cyclic analog of neuropeptide Y, [Ahx5-24, gamma-Glu2-epsilon-Lys30] NPY (YESK-Ahx-RHYINKITRQRY; Ahx, 6-aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin-stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5-24] NPY (YPSK-Ahx-RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2-receptor-expressing cell line was slightly reduced, compared to the linear peptide [Ahx5-24] NPY, however inhibition of cAMP accumulation was even more efficient. Unlike the linear peptide [Ahx5-24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor-expressing SK-N-MC cells, as well as the loss of capability of signal transduction, suggest that only Y2-mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY-(13-36) peptide, which has been used to determine Y2 selectivity so far.


Complete down-regulation of low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2 by beta-migrating very-low-density lipoprotein and non-lipoprotein cholesterol. Different cellular regulatory pools of cholesterol. Regulation of low-density-lipoprotein-receptor activity by low-density lipoprotein (LDL), cholesteryl-ester-rich beta-migrating very-low-density lipoprotein (beta-VLDL) and non-lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2. Competition studies indicate that LDL and beta-VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or beta-VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high-affinity association of LDL and 26% of the high-affinity degradation of LDL, while with beta-VLDL a complete down regulation of the LDL-receptor activity is observed. Preincubation of Hep G2 cells with beta-VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acyl-coA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6-fold. The more effective down regulation of LDL receptors by beta-VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with beta-VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL-receptor activity. Non-lipoprotein cholesterol, however, caused a complete down regulation of LDL-receptor activity at even lower extracellular cholesterol concentrations than with beta-VLDL. The complete down regulation of LDL receptors by non-lipoprotein cholesterol is not accompanied by a significant increase in acyl-coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6-fold. It is suggested that the effectiveness of non-lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor. The observed complete down regulation of the LDL receptor by beta-VLDL may be responsible for the cholesterol-rich-diet induced, complete down regulation of LDL-receptor-mediated clearance of LDL in vivo.


Testicular carcinoma with inferior vena cava thrombosis extending into the right atrium treated with chemotherapy and anticoagulation. A 34-year-old male with pulmonary emboli and thrombosis of the inferior vena cava extending into the right atrium was found at presentation to have a mixed seminoma and embryonal cell testicular carcinoma with high-volume retroperitoneal disease and visceral metastases. The patient was free of disease 19 months after treatment with combination chemotherapy and anticoagulation followed by resection of the residual mass. We could not find any previous report of a patient with bulky retroperitoneal disease and vena cava thrombosis successfully treated with chemotherapy without vena cava resection.


A comparison of the effect of lisinopril and hydrochlorothiazide on electrolyte balance in essential hypertension. The effects of lisinopril 10-20 mg or hydrochlorothiazide 25-50 mg (each given once daily) on blood pressure, serum sodium, potassium and magnesium concentrations, total body potassium and urinary cation excretion were compared in a group of hypertensive patients using a double blind randomised crossover design. Each active treatment phase lasted six weeks and a total of sixteen patients completed the study. Both lisinopril and hydrochlorothiazide produced clinically significant decreases in blood pressure. However, lisinopril treatment produced a mean reduction of 14 mmHg in sitting diastolic pressure compared with a 7 mmHg reduction for hydrochlorothiazide treatment. This difference was statistically significant. The decrease in the concentration of serum potassium during hydrochlorothiazide treatment was greater than that during lisinopril treatment (0.53 vs 0.01 mmol.1). The absolute value of serum potassium was significantly lower on hydrochlorothiazide than on lisinopril therapy. Neither treatment had an effect on serum magnesium concentrations, nor was there any significant effect of either treatment on urine volume or urinary excretion of sodium, potassium or magnesium. There was a trend towards increased total body potassium concentration on lisinopril compared with a decrease in total body potassium on hydrochlorothiazide. However, this difference was just outside the range of statistical significance. Both treatments were equally well tolerated. The results indicate slight superiority of lisinopril over hydrochlorothiazide with regard to control of diastolic blood pressure with a better effect on overall electrolyte balance.


Coexpression of the receptor-associated protein gephyrin changes the ligand binding affinities of alpha 2 glycine receptors. The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein, whose ligand binding alpha subunit occurs in several isoforms in the mammalian central nervous system. Here we show that coexpression of the GlyR-associated protein gephyrin changes the agonist and antagonist binding affinities of GlyRs generated by alpha 2 subunit expression in 293 kidney cells. Thus, a receptor-associated protein modifies the functional properties of a neurotransmitter receptor. This may contribute to an optimization of the postsynaptic neurotransmitter response.


Expression pattern of the activin receptor type IIA gene during differentiation of chick neural tissues, muscle and skin. To elucidate target cells of activins during embryogenesis we isolated cDNAs of chick activin receptor type II (cActR-II) and studied expression patterns of the cActR-II gene by in situ hybridization. Transcripts of cActR-II were observed in neuroectoderm developing to spinal cord, brain and eyes, in surface ectoderm differentiating to epidermis, and in myotomes differentiating to muscles. The expression patterns of cActR-II suggest that activin and its receptor are involved in differentiation of chick neural tissues, muscle and skin after inducing the dorsal mesoderm.


Xanthine oxidase/hydrogen peroxide generates sulfur trioxide anion radical (SO3.-) from sulfite (SO3(2-)). In the presence of hydrogen peroxide (H2O2), xanthine oxidase has been found to catalyze sulfur trioxide anion radical (SO3.-) formation from sulfite anion (SO3(2-)). The SO3.- radical was identified by ESR (electron spin resonance) spin trapping, utilizing 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) as the spin trap. Inactivated xanthine oxidase does not catalyze SO3.- radical formation, implying a specific role for this enzyme. The initial rate of SO3.- radical formation increases linearly with xanthine oxidase concentration. Together, these observations indicate that the SO3.- generation occurs enzymatically. These results suggest a new property of xanthine oxidase and perhaps also a significant step in the mechanism of sulfite toxicity in cellular systems.


Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation. High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.


Hemin stimulation of cAMP production in human lymphocytes. Hemin stimulates cAMP production in human peripheral blood mononuclear cells (PBMC). The kinetics are similar to that of hormone-induced cAMP generation, namely a rapid effect followed by a desensitization phase. Several experimental findings suggest that prostaglandins do not mediate this effect. First, macrophage depleted T and B cells purified by erythrocyte-rosetting were as responsive as unfractionated PBMC to hemin. Second, indomethacin, an inhibitor of prostaglandin synthesis, and meclofenamate, a prostaglandin E2 receptor antagonist, had no effect on hemin stimulated cAMP production. In addition, propranolol, a beta-adrenergic receptor antagonist, had no effect on hemin-stimulated cAMP production. We also examined structural analogues of hemin. Among the metalloporphyrins (Fe, Ni, Co, Zn and Sn) and protoporphyrin IX tested only hemin (Fe-protoporphyrin) was active in stimulating cAMP production. No correlation was found between the ability of metalloporphyrins to stimulate cAMP production and their ability to generate H2O2. The data indicate that hemin stimulates cAMP production by directly affecting lymphocytes and that prostaglandins do not mediate this effect.


Potential protective effect of vitamin C on carcinogenesis caused by nitrosamine in drinking water: an experimental study on Wistar rats. In an experiment with Wistar rats the question of whether oral vitamin C application has a protective effect in liver carcinogenesis caused by N-nitrosodiethylamine in drinking water (n = 104) was studied. When comparing four groups (without carcinogen, with carcinogen and carcinogen plus vitamin C alternately and concomitantly) significant statistical differences in the linear regression were noted. Ninety per cent of the rats developed hepatocellular carcinomas when only carcinogen was administered whereas the rate with additional administration of vitamin C alternately and concomitantly was 68% and 55%, respectively. Without carcinogen no tumour developed. The results suggest that vitamin C may delay the development of liver cancer upon oral administration of N-nitrosodiethylamine. The explanation for this fact is still hypothetical.


The place for conservative treatment in the management of Paget's disease of the nipple. Four cases of Paget's disease confined to the nipple are presented. Three of them were treated conservatively by local radiotherapy and the fourth was treated by mastectomy. Histopathological examination of the breast in the last case failed to show any evidence of in situ or invasive carcinoma. The first three patients are well and without evidence of recurrence 3 to 5.5 years after treatment. Could conservative treatment in the form of local excision or radiotherapy offer an alternative to mastectomy? A discussion on the place of conservative treatment is presented.


Benign mixed salivary-type tumour of the breast. A case of benign mixed salivary-type tumour of the breast is described. This is a rare neoplasm, only 20 cases having been reported to date, characterized by a mixture of epithelial and mesenchymal components, as in similar tumours occurring in the salivary glands and skin. Because this tumour frequently simulates carcinoma clinically, mammographically and histologically, familiarity of both the surgeon and pathologist with this lesion is essential, to avoid the overdiagnosis of malignancy, unfortunately initially made in nearly 50% of previously reported cases.


Human papillomavirus detection from the female genital tract: combined vs. separate scrape methods. Because genital human papillomavirus (HPV) infections tend to be multifocal, it was studied how effective one combined specimen is in detecting HPV-DNA from the lower female genital tract. The study population consisted of 50 patients referred to a colposcopy clinic for a suspected condylomatous and/or dysplastic lesion. From half of the patients, a separate scrape from the cervix, vagina and vulva was taken first followed by a combined scrape representing all the genital sites, and from the other half, vice versa. HPV-DNA (types 6, 11, 16 and 18) was identified using the AffiProbe hybridisation test. Thirty-six specimens collected from 17 patients were positive for HPV-DNA. A multifocal infection was demonstrated in at least 11/17 (65%) HPV-DNA positive patients. The combined scrape was the most informative specimen, revealing 75% of all HPV-DNA-positive patients. It was concluded that HPV-DNA can reliably be detected from the female genital tract in a simple way from one combined specimen.


Dual functional role of membrane depolarization/Ca2+ influx in rat pancreatic B-cell. Transient exposure of rat pancreatic B-cell to 50 mM K+ ([K+50]) makes exocytosis unresponsive to further depolarization, i.e., stimulation with 100 mM K+ or 1 uM glyburide, which closes the ATP-sensitive K+ (K+ATP) channel, simultaneously with [K+50] does not produce any greater insulin secretion compared with [K+50] alone. In sharp contrast, 16.7 mM glucose ([G16.7]) applied simultaneously with [K+50] elicits an insulin response markedly greater than that produced by [K+50] alone, which is not attenuated by 100 uM diazoxide, an inhibitor of K+ATP channel closure. [G16.7]-induced insulin secretion at the basal K+ concn of 4.7 mM was greatly (93%) suppressed by 100 uM diazoxide. Insulin secretion induced by [K+50] plus [G16.7] ([K+50 + G16.7]) was markedly suppressed (70%) by 1 uM nifedipine, a Ca(2+)-channel blocker and was completely abolished by 2 mM 2-cyclohexen-1-one, which reportedly decreases reduced glutathione level and blocks glucokinase. This finding indicates that insulin release induced by [K+50 + G16.7] is not due to leakage produced by toxic stimuli but to activation of exocytosis. When graded concentrations (25 and 50 mM) of K+ were applied simultaneously with [G16.7] in the presence of 100 uM diazoxide, insulin response was clearly dependent on K+ concentration, indicating that the physiological range of membrane depolarization also activates the glucose-responsive effector. Membrane depolarization/Ca2+ influx directly stimulates hormone exocytosis on one hand and activates the K+ATP channel-independent glucose-responsive effector or effectors on the other in the B-cell. The nature of the glucose-responsive effector or effectors remains to be established.


Pioglitazone increases insulin sensitivity by activating insulin receptor kinase. A new oral agent, 5-[4-(2-(5-ethyl 12-pyridyl)ethoxy]- benzoyl]-2,4-thiazolidinedione (pioglitazone), has been developed for treatment of non-insulin-dependent diabetes mellitus (NIDDM). This agent increases insulin sensitivity in vivo in genetically obese Wistar fatty rats. Administration of the agent (3 mg/kg/day) for 10 days to the rats ameliorated hyperglycemia and hyperinsulinemia, indicating that it decreased insulin resistance. To clarify the mechanism of the drug to increase insulin sensitivity, we examined insulin binding and kinase activity of insulin receptors from muscles of both untreated and treated rats. Pioglitazone treatment did not change insulin binding in Wistar fatty rats but increased insulin-stimulated autophosphorylation of insulin receptors to 78% over the level in the control but not the basal state. Kinase activity toward exogenous substrate, poly Glu4Tyr1, was also increased to 87% over the level of untreated control obese rats. In contrast, in lean rats, pioglitazone treatment did not increase autophosphorylation and kinase activity toward exogenous substrates. To further elucidate the mechanism, we incubated insulin receptors with the agent and measured kinase activity. Incubation of solubilized receptors with the agent did not increase kinase activity. However, the receptors from IM-9 cells, which were incubated with 10(-8) M pioglitazone for 7 days, showed a 46% increase over the control in insulin-stimulated autophosphorylation and kinase activity. These results suggested that pioglitazone increased insulin sensitivity in part by activating kinase of the receptors through indirect effect on insulin receptors and that the drug may have useful benefits in insulin resistance of NIDDM.


[The value of nuclear magnetic resonance tomography in diagnosing invasive trophoblast disease]. 13 patients between 19 and 35 years, 12 of them with histologically verified trophoblastic disease, were examined by MRI, to prove the diagnostic value of this method. In line with the excellent results of MRI described in the literature with respect to the differentiation of tissue characterisation, vascularisation and localisation of tumours, the response to chemotherapy was evaluated comparatively to HCG-titres (5 patients). The results showed, that a decrease of HCG-titres correlated with significant tumour reduction. MRI was performed with 1.5 Tesla machines. Axial, sagittal and coronal images of pelvis and abdomen in T1, proton density and T1 sequences, one after administration of Gd-DTPA, were carried out. Except for one case with decreasing titres, GTP was proven by MRI. The tumour showed a remarkable lack of homogeneity and signal increase in 7 cases in T2-weighted images. In one case, the tumour showed a marked signal enhancement after applying Gd-DTPA, compared with the normal myometrium. In 5 cases, destruction of the zonal anatomy of the uterus and dilatation of subserosal uterine and pelvis veins were found. The uterus was larger in size than expected for the duration of gravidity. In correlation with the clinical examination and HCG titres, MRI has the potential of multiplanar imaging and signal characterisation of tissue, and is therefore useful to evaluate the tumour extent and to detect residuals. Gradient echo-technique and the use of contrast media will reduce the duration of examination, but more experience is required.


[Clinical significance of N-14 antibody (ELISA) in diagnosis of non-A, non-B liver disease]. In 1987 Chiron Co. in USA and Arima et al. in Japan reported successful isolation of clones coding peptides specific for non-A, non-B hepatitis infection. Hepatitis C virus (HCV) genome reported by the Chiron is supposed to have structural proteins of core, matrix, envelope and non-structural proteins of NS 1-5 from 5' to 3' end. C-100 antibody of which antigen is derived from the NS 3-4 region of the HCV genome is used generally, it is not sufficient to diagnose hepatitis C. On the other hand, the nucleotide and amino acid sequences in the epitope of N-14 clone established by Arima et al. have homology to those of core region of the HCV genome. Usually there are some mutation in sequences of HCV genome, but there is little mutation in 5' non-coding region and core protein area. Therefore we could diagnose hepatitis C more exactly by using N-14 antibody. In the present study N-14 antibody was determined in 871 healthy subjects and 285 cases with liver diseases by an ELISA. 1.6% of healthy subjects and 70-75% of cases with non-A, non-B chronic liver diseases are positive for N-14 antibody. In acute non-A, non-B hepatitis, 33.3% of sporadic cases and 75.0% of post-transfusion cases are positive for the test. As only 2 of 100 cases with other liver diseases are positive for the test, the N-14 antibody test seems to be highly sensitive and specific for the non-A, non-B hepatitis virus infection. These 1156 cases were tested for the C-100 antibody as well. No much difference between the results by using these tests, 15-20% of discrepancy between the tests, has been observed. In conclusion hepatitis C can be diagnosed exactly by using N-14 antibody as well as by C-100 antibody. In addition for more exact diagnosis of hepatitis C, tests by N-14 antibody in combination with C-100 antibody will be prefered to determine anti HCV.


In vitro stability of the inhibition of serum converting enzyme by fosinopril. With captopril, it has been shown that an erroneous measurement of serum angiotensin converting enzyme (ACE) can be induced by the dissociation of the inhibitor-ACE complex during long-term storage. We have studied the possible dissociation of the fosinopril-ACE complex during the storage of serum samples from healthy male volunteers given a single dose of fosinopril. Serum samples were collected from 5 volunteers, 5 min before, then 4 and 24 h after a unique oral dose of 10 mg fosinopril. ACE activity was measured by a colorimetric and a fluorimetric assay during the hour following the sampling (day 0) and after 21 or 61 days of storage at -20 or -196 degrees C. The degree of ACE inhibition measured in vitro in fresh serum samples differed according to the technique used. Fosinopril has a long-lasting effect with 80% inhibition 24 hours after drug administration. Storage at -20 and -196 degrees C induced a significant decrease in the degree of inhibition measured with the colorimetric method. With the fluorimetric method, a decrease in ACE inhibition was only observed after storage at -20 degrees C but not at -196 degrees C.


Hepatoid adenocarcinoma of the papilla of Vater. An adenocarcinoma of the papilla of Vater showing hepatoid differentiation similar to that described for hepatoid adenocarcinoma of the stomach is reported. The tumor was a poorly differentiated adenocarcinoma with clear cells containing occasional hyaline droplets and exhibiting bile secretion. Alpha-fetoprotein (AFP), alpha 1-antitrypsin (alpha 1-AT) and carcino-embryonic antigen (CEA) were demonstrated by immunoperoxidase staining. This appears to be the first report of a hepatoid adenocarcinoma at this location.


Excitatory amino acid regulation of neuronal functions. Excitatory amino acids (EAAs), usually glutamate, activate the greatest excitatory system in the vertebrate brain. EAAs mediate synaptic transmission and control the stability and efficiency of synaptic connections. Moreover, EAAs provide the postsynaptic neurone with chemical cues for survival and maturation. Improper activation of EAA receptors can lead to neurotoxicity and be involved in the pathogenesis of certain neurological diseases.


Carbachol-evoked suppression of excitatory neurotransmission in guinea-pig olfactory cortex slices is unlikely to involve an M4-muscarinic receptor subtype. Depression of the electrically-evoked surface-negative field potential (N-Wave) by bath-superfusion of carbachol was measured in guinea-pig olfactory cortex slices maintained in vitro. The possibility that this response, previously proposed to be mediated via a presynaptic M1: muscarinic receptor, might in fact be due to M4 receptor activation, was investigated by testing the effectiveness of himbacine (a proposed M4-selective antagonist) on our cortical preparation. Himbacine (100 nM-1 microM) had no effect on the N-wave potential alone, but it induced a clear competitive-type inhibition of carbachol effects. Schild plot analysis (regression slope constrained to unity) of pooled data yielded a pA2 value of 7.2 for this antagonist (n = 7 slices). This value accords more with that expected for the interaction of himbacine with M1 receptors (approximately 7.2) than with functionally expressed M4 receptors (approximately 8.5-8.5). We therefore conclude that M4-type muscarinic receptors are unlikely to be involved in mediating this presynaptic carbachol response.


Membrane potentials in retinal capillary pericytes: excitability and effect of vasoactive substances. Retinal capillary pericytes are believed to have a contractile function and to regulate retinal blood flow at the microvascular level. Membrane potential is an important control element for contractility in smooth muscle cells. In the present study, bovine retinal capillary pericytes have been grown in tissue culture and membrane potentials have been measured using glass microelectrodes. Resting potentials averaged -31 +/- 7 mV (n = 203). Relative K+ conductance was low, with a transference number for K+ of 0.16. Readdition of K+ to K(+)-depleted cells transiently hyperpolarized the membrane potential, probably by stimulating the electrogenic Na+/K+ transport. Repetitive spike-like depolarizations (action potentials) were induced by stimulating the Na+/K(+)-ATPase, by applying norepinephrine (10(-5) mol/l), and by adding 10 mmol/l Ba2+. These action potentials depended on the presence of extracellular Ca2+ and were inhibited by the Ca2+ antagonist nifedipine (10(-6) mol/l). Norepinephrine (10(-5) mol/l) depolarized the membrane by 7.4 +/- 3.5 mV (mean +/- SD, n = 49). This response was blocked by the alpha 1-antagonist prazosin (10(-5) mol/l). Histamine also led to a membrane depolarization of 8.6 +/- 2.8 mV (n = 49), which could be inhibited by the H1-antagonist diphenhydramine. Endothelin (10(-7) mol/l), vasopressin (10(-6) mol/l), and acetylcholine (10(-4) mol/l) had no major effects on membrane potential. The conclusion is that retinal capillary pericytes are excitable cells and react to several vasoactive substances.


Synergistic uveitic effects of tumor necrosis factor-alpha and interleukin-1 beta. Tumor necrosis factor (TNF) and interleukin-1 (IL-1), cytokines with multiple, overlapping biologic activities, have been shown to interact synergistically in nonocular tissues. To test the hypothesis that coinjection of TNF and IL-1 interact synergistically in the eye, low, marginally inflammatory doses of human recombinant TNF-alpha (4000 U), IL-1 beta (40 U), and TNF-alpha+IL-1 beta (TNF-alpha/IL-1 beta) were injected into the vitreal chamber of the rabbit eye, and inflammation was assessed at 6, 24, 48, and 168 hr post-cytokine injection. TNF-alpha/IL-1 beta induced an anterior uveitis that was barely detectable at 6 hr, increased at 24 hr, peaked at 48 hr, and largely resolved by 168 hr. Synergy was observed for infiltration of inflammatory leukocytes into aqueous humor at 24 and 48 hr and for protein and prostaglandin E levels in aqueous humor at 48 hr. Based upon protein levels in vitreous humor, TNF-alpha/IL-1 beta also induced a posterior uveitis. This posterior uveitis was not apparent until 48 hr and then increased significantly at 168 hr. At 48 and 168 hr, the effects of TNF-alpha/IL-1 beta on protein levels in vitreous humor were consistent with a synergistic interaction. Results of separate experiments using higher dose combinations of TNF-alpha/IL-1 beta and a longer time course suggested that the effects of TNF-alpha/IL-1 beta on the blood vitreous barrier persisted beyond 168 hr. The results of this study support the hypothesis that TNF-alpha and IL-1 beta interact synergistically when injected into the rabbit eye.(ABSTRACT TRUNCATED AT 250 WORDS)


A rabbit corneal epithelial cell line expresses functional platelet-derived growth factor beta-type receptors. Platelet-derived growth factor (PDGF) is a family of three isoforms termed PDGF-AA, PDGF-AB, or PDGF-BB that induce proliferation in various mesenchymal cells. A rabbit corneal epithelial cell line (SIRC) was chosen to study the effects of the three isoforms of PDGF. These cells express approximately 43,000 PDGF beta-type receptors and less than 2800 alpha-type receptors on their surface. Thus, only PDGF-BB and -AB led to a transient dose-dependent rise in cytosolic free calcium with an effective dose for 50% of cells of 5 ng/ml. The PDGF-induced calcium signal is largely independent of the presence of extracellular calcium.


Inflammatory effects of continuous-wave neodymium: yttrium aluminum garnet laser cyclophotocoagulation. The uveal inflammatory response was studied in 31 rabbits treated unilaterally with neodymium: yttrium aluminum garnet (Nd:YAG) cyclophotocoagulation. Fifteen applications of 3.5-J energy were delivered to the dorsal and ventral perilimbal sclera using a contact continuous-wave system. On days 1, 3, 8, and 15, the inflammatory effects were assessed. Peak levels of aqueous humor protein (11 +/- 3 mg/ml), prostaglandin E2 (8.9 +/- 3.0 ng/ml), leukocytes (205 +/- 113/microliters), and iris-ciliary body myeloperoxidase activity (6.32 +/- 1.4 U/mg protein) occurred on day 3 and rapidly decreased between days 7 and 15. Vitreal protein levels also peaked at day 3 but remained elevated through day 15 (3.8 +/- 1.3 mg/ml). By contrast, aqueous erythrocytes were most numerous (22,614 +/- 10,517/microliters) on day 8. Levels of leukotriene B4 remained low in all eyes at all intervals. Correlative histologic changes were ciliary coagulation necrosis, severe vascular congestion, and a predominantly mononuclear inflammatory cell infiltrate. These data suggest that Nd:YAG cyclophotocoagulation in rabbits induces a relatively mild inflammatory response that is associated with significant vascular compromise. Although these observations may not be analogous to the situation in the human eye, they may provide a model with which to compare the relative effects of different treatment parameters to help establish the optimum protocol.


Response of metastatic adenoid cystic carcinoma and Merkel cell tumor to high-dose melphalan with autologous bone marrow transplantation. Two patients with metastatic spread of unusual tumors responded to treatment with high-dose Melphalan and autologous bone marrow transplant. One patient had adenoid cystic carcinoma of a minor salivary gland and the other had Merkel cell tumor of the scalp. Both patients had undergone prior surgery and radiotherapy, but later relapsed with distant metastases. Both patients had progression of their disease despite conventional and salvage chemotherapy. Treatment with high-dose Melphalan and autologous bone marrow transplant resulted in partial responses for both patients. High-dose Melphalan should be considered for therapy earlier in the course of patients with these unusual cancers.


Hepatic cancers and their response to chemoembolization therapy. Quantitative image-guided 31P magnetic resonance spectroscopy. Hepatic embolization combined with intra-arterial administration of cytostatic drugs (chemoembolization) is frequently used to treat primary and metastatic cancers to the liver. Quantitative phosphorus-31 magnetic resonance spectroscopy (31P MRS) was used to assess the metabolic state of hepatic cancers and their metabolic response to chemoembolization. Fifteen localized 31P MRS studies were performed on five patients with liver tumors. Thirteen healthy volunteers served as controls. Metabolite ratios and molar metabolite concentrations were calculated. Untreated hepatic tumors, relative to normal controls, showed elevated phosphomonoester/adenosine triphosphate (PME/ATP) ratios, reduced concentrations of ATP and inorganic phosphate (Pi), and normal phosphodiester (PDE) concentrations. As an acute response to chemoembolization, ATP, PME, and/or PDE concentrations diminished, whereas Pi concentrations increased or stayed relatively constant. Long-term follow-up after chemoembolization showed decreased PME/ATP and increased ATP concentrations in the absence of changes on standard magnetic resonance and computed tomographic images. These preliminary spectroscopic data suggest that quantitative 31P MRS can be successfully used to monitor directly metabolic response to hepatic chemoembolization.


Synthesis of potential 99mTc nitrido tumor imaging disposition in mice. Several cationic or neutral technetium-nitrido complexes of Schiff bases [for which 5-methyl 3-(2-hydroxyphenyl methylene) dithiocarbazate (L1) was a prototype], bis-aminoethanethiol (BAT-TM) and macrocyclic amines were prepared. We report here, the synthesis and isolation of these TcNLn complexes from reaction mixtures by high pressure liquid chromatography or sephadex G25 column. In vivo tissue distribution studies were performed on tumor bearing mice (B16, EMT6, 3LL) after i.v. injection of 10-20 microCi of TcNLn. Although pharmacokinetic differences appeared between the three studied ranges, we did not obtain proof of any particular specificity to tumor tissues. Nevertheless, the complexes were very stable and some of the ligands could be used as chelators after linking side chains or groups inducing specific tumor localization.


Optimization of [11C]CO2 trapping efficiencies from nitrogen gas streams. Three new systems were developed for trapping [11C]CO2 from high flow rate nitrogen gas streams and the trapping efficiency was determined for each trap design. The results of this study indicate that both the stainless steel shot and stainless steel frit systems yield high trapping efficiencies under the range of flow rates evaluated (400-3600 sccm) and may prove to be alternatives to the traditional stainless steel/copper coiled tubing or molecular sieve trapping systems in common use.


Identification of the aadB gene coding for the aminoglycoside-2"-O-nucleotidyltransferase, ANT(2"), by means of the polymerase chain reaction. The polymerase chain reaction (PCR) was used to identify the gene encoding the aminoglycoside-2"-O-nucleotidyltransferase, ANT(2"). Two primers, delineating a DNA fragment of 188 bp, and a specific probe within this fragment were constructed, based on the nucleotide sequence of the aadB gene encoding this enzyme. Reference strains producing different aminoglycoside-modifying enzymes were used to evaluate the specificity and the sensitivity of the test. Strains producing the ANT(2") enzyme showed the expected 188 bp DNA fragment after amplification. The oligonucleotide primers did not interact with genes encoding other aminoglycoside-modifying enzymes. Evaluation of a one-step single colony technique demonstrated that it was an acceptable alternative to the classic PCR test. The PCR method was used successfully to detect the presence of the aadB gene in 17 gentamicin-resistant clinical isolates.


Mitochondrial targeting signal of rat liver monoamine oxidase B is located at its carboxy terminus. Monoamine oxidase B, a typical intrinsic protein of the outer mitochondrial membrane, has an uncleavable targeting signal and is inserted into the membrane without proteolytic maturation. To investigate the region responsible for targeting the enzyme to the outer mitochondrial membrane, various mutated proteins were expressed in cultured mammalian cells, and the distributions of the expressed proteins were analyzed by immunofluorescence microscopy and subcellular fractionation. Deletion of the carboxy-terminal 28 amino acids of monoamine oxidase B abolished the transfer of the enzyme to mitochondria, while the deletion of the amino-terminal 55 amino acids had no effect on the transfer to mitochondria. The existence of the targeting signal at the carboxy-terminal portion of the enzyme was confirmed by using hybrid proteins in which the amino- or carboxy-terminal portion of the enzyme was fused to the hydrophilic portion of cytochrome b5. The fused protein with the carboxy-terminal 29 amino acid residues of monoamine oxidase B was localized in mitochondria, whereas that with 10 amino acids remained in the cytoplasm. These results indicate that the targeting signal of monoamine oxidase B is present within its carboxy-terminal 29 amino acid residues.


Phosphatidyl inositol 4,5-bisphosphate-specific phospholipase C in bovine rod outer segment membranes. Polyphosphoinositide-specific phosphodiesterase (phospholipase C) activity against phosphatidylinositol 4,5-bisphosphate has been examined in disrupted bovine retinal rod outer segments. The enzyme was strictly modulated by free calcium ion concentration and maximally activated at 10(-5) M Ca2+ (91 +/- 4 nmoles phosphatidylinositol 4,5-bisphosphate hydrolyzed/min/mg of protein). Guanine nucleotides did not affect in vitro phospholipase C activity either in the presence or absence of light, carbachol or epinephrine. The pH optimum at 10(-5) M Ca2+ in the presence of sodium deoxycholate was 6.5. The enzyme of bovine rod outer segments was concluded to be indirectly regulated by the phototransduction events.


Thiobacillus novellus cytochrome c oxidase contains one heme alpha molecule and one copper atom per catalytic unit. The minimal structural unit of cytochrome c oxidase purified from Thiobacillus novellus was composed of one molecule each of two subunits with molecular masses of 32 and 23 kDa, respectively, and the unit had one molecule of heme a and one atom of copper. In the presence of n-octyl-beta-D-thioglucoside, the oxidase existed as the monomeric form of the unit, while it occurred as the dimeric form of the unit in the presence of Tween 20. The monomeric form showed an active cytochrome c oxidizing activity and reduced molecular oxygen to water with ferrocytochrome c. Namely, it has been shown that the bacterial cytochrome c oxidase with one heme a molecule and one copper atom per molecule can catalyze oxidation of ferrocytochrome c with concomitant reduction of molecular oxygen to water.


Comparison of extracellular matrix-degrading activities between 64-kDa and 90-kDa gelatinases purified in inhibitor-free forms from human schwannoma cells. Two kinds of gelatinases (or type IV collagenases), 90-kDa and 64-kDa gelatinases, were purified in a tissue inhibitor of metalloproteinases (TIMP)- or TIMP-2-free form from the serum-free conditioned medium of human schwannoma YST-3 cells, and their activities on extracellular matrix proteins were compared. Sequential chromatographies on a gelatin-Sepharose column, an LCA-agarose column, and a gel filtration column in the presence of 5 M urea yielded 600 micrograms of the 64-kDa enzyme and 45 micrograms of the 90-kDa enzyme from 2.8 liters of the conditioned medium. The purified enzymes showed high gelatinolytic activities without activation by p-aminophenyl mercuric acetate (APMA), indicating that 5 M urea used in the final chromatography not only dissociated the inhibitors from the progelatinases but also activated the proenzymes. The inhibitor-free gelatinases showed a much higher activity than the APMA-activated inhibitor-bound enzymes. The specific activity of the 90-kDa enzyme was nearly 25 times higher than that of the 64-kDa enzyme. The 90-kDa gelatinase hydrolyzed type I collagen as well as native and pepsin-treated type IV collagens at 30 degrees C, while at 37 degrees C it potently hydrolyzed types I, III, and IV collagens but not fibronectin or laminin. The 64-kDa gelatinase showed a similar substrate specificity to that of the 90-kDa enzyme, except that it did not hydrolyze type I collagen and native type IV collagen at 30 degrees C.


Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase. The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.


Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic. The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C. At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 microM forskolin, and 3 mM 8Br-cAMP were intact. Re-warming above 32 degrees C restored CT-induced Isc. Preincubating monolayers for 30 min at 37 degrees C before cooling to 15 degrees C overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited. These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells. We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.