Fine-needle aspiration biopsy of small round blue cell tumors of childhood.

One hundred twenty-eight palpable and deep-seated fine-needle aspiration biopsies (FNAB) were done on pediatric patients at James Whitcomb Riley Hospital for Children and Indiana University Hospital between 1985 and 1988. During that 4-year period, 71 (56%) benign and 49 (38%) malignant diagnoses were made. Only eight (6%) of the FNAB were considered inadequate. Thirty-nine (80%) of the malignant aspirates were small round blue cell tumors of childhood (SRBCT). The SRBCT consisted of 21 (54%) lymphomas, 7 (18%) Ewing's sarcomas, 3 (8.5%) neuroblastomas, 3 (8.5%) rhabdomyosarcomas, 2 (5.0%) medulloblastomas, 2 (5.0%) Wilms' tumors, 1 (3.0%) retinoblastoma, and 1 (3%) granulocytic sarcoma. Fifteen (38%) of the SRBCT aspirates were obtained to render a primary diagnosis and 24 (62%) documented recurrence. Various combinations of electron microscopy, immunocytochemistry, and other special stains were used to confirm the diagnosis in 11 (28%) cases. These cases consisted of five lymphomas, two rhabdomyosarcomas, two Ewing's sarcomas, one neuroblastoma, and one granulocytic sarcoma. The technique of FNAB is a successful diagnostic tool for documenting primary and recurrent SRBCT in a pediatric population.


Xenotransplantation of alpha-fetoprotein-producing gastric cancers into nude mice. Characteristics and responses to chemotherapy.

Three human alpha-fetoprotein (AFP)-producing gastric cancers (AFPGC) were xenotransplanted into the lateral abdominal wall of nude mice. Two tumors were established and passed over ten generations. These tumors retained their ability to secrete AFP and their characteristic hepatoid features microscopically. Serum levels of AFP in the mice were elevated as the tumors grew. Through serial transplantation, the degree of differentiation was not altered. Neither local invasion nor distant metastasis were encountered during the observation periods. Both strains had an aneuploid pattern of DNA by flow cytometric examinations. The responses of these tumors to five chemotherapeutic agents were investigated using various doses. The high AFP-titer strain (AFPGC-2) showed a marked regression or suppression of tumor growth after administration of both mitomycin C (MMC) and cisplatin (CDDP). The low-titer strain (AFPGC-1) had substantial sensitivity only to MMC. The growth of both tumors was not suppressed by 5-fluorouracil, doxorubicin, or epirubicin. These findings suggest that the characteristics of AFPGC are preserved in the xenograft model of nude mice. In addition, MMC and CDDP may be active to some extent against this rare, but highly malignant cancer.


Treatment of unresectable primary liver cancer with intrahepatic fluorodeoxyuridine and mitomycin C through an implantable pump.

Ten patients with unresectable primary liver cancer, eight of whom had elevated serum alpha-fetoprotein levels, were treated with intrahepatic fluorodeoxyuridine (FUDR) and mitomycin C administered through an implantable pump. Four patients had a partial response, and two had a minor response. The median survival from initiation of treatment was 14.5 months (range, 2 to 32 months), with patients receiving therapy for a median of 11.2 months. In general, the therapy was well tolerated; only one patient had treatment-related biliary sclerosis. In conclusion, the combination of intrahepatic FUDR and mitomycin C was an effective palliative regimen for unresectable primary liver cancer, even in the presence of elevated serum alpha-fetoprotein levels. Further studies are needed to confirm these findings and compare this regimen with other methods of treatment for hepatocellular carcinoma.


Differential diagnosis of borderline and invasive serous cystadenocarcinomas of the ovary by computerized interactive morphometric analysis of nuclear features.

Whether borderline serous tumors of the ovary can be differentiated from invasive serous cystadenocarcinomas by morphometric analysis of nuclear features of the neoplastic epithelium was examined. Multiple descriptors extracted from nuclear tracings and intranuclear gray-level analysis of argyrophilic nucleolar organizer regions were evaluated in 11 borderline and 18 invasive tumors using computerized interactive morphometric analysis. These descriptors included: nuclear area and perimeter; number, area, and perimeter of argyrophilic nucleolar organizer regions; standard deviations of these findings; and a size distribution of nuclear areas in discrete classes. Multivariate statistical analysis showed the internal consistency of the two groups in terms of physical descriptors and the discriminating value of the parameters of nuclear size and pleomorphism (independently of nucleolar organizer region parameters). The results indicated that interactive morphometric analysis of nuclear features, combined with appropriate statistical methods, could be used to distinguish between these tumors.


Enhancement of chemotherapeutic effects by recombinant human granulocyte colony-stimulating factor on implanted mouse bladder cancer cells (MBT-2).

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered intraperitoneally in combination with multidrug chemotherapy using methotrexate (M), vinblastine (V), doxorubicin (A), and cisplatin (C, or for the combination, MVAC) to C3H/He mice (5-week-old females) after experimental carcinoma, MBT-2, a transplantable transitional cell carcinoma of the urinary bladder had been implanted. The effects of therapy were studied. The animal groups consisted of: (1) control (no drug administration), (2) rhG-CSF (100 micrograms/kg/d, from days 8 through 42 after MBT-2 implantation, except for the days when MVAC was administered), (3) high-dose MVAC (2 mg/kg of M, 0.2 mg/kg of V, 2 mg/kg of A, and 4 mg/kg of C once a week for 3 weeks), (4) low-dose MVAC (one-quarter of the high dose), (5) high-dose MVAC with rhG-CSF, and (6) low-dose MVAC with rhG-CSF. In an in vitro system, rhG-CSF did not show any effect on the proliferation of MBT-2 cells or exert any influences on A's tumor proliferation-suppressing action on MBT-2. However, in an in vivo system, concomitant administration of rhG-CSF significantly enhanced the tumor-suppressing effect of the MVAC therapy, as did rhG-CSF alone. The greatest effect was observed in the group receiving high-dose MVAC plus rhG-CSF. These result suggested that rhG-CSF-stimulated granulocytes may exert antitumor activity on tumor cells severely damaged by chemotherapeutic agents at a relatively high concentration. The survival rate was improved to some degree even by administration of rhG-CSF alone. Although further study is required to elucidate the action mechanism of rhG-CSF, these results suggest that rhG-CSF may be useful clinically to enhance the activity of antitumor agents and not only through its ability to alleviate granulocytopenia or prevent its development.


Confocal microscopic and other observations on the distal end of the thick limb of the human loop of Henle.

Various antibodies and lectins were used in a histological study of the human renal tubule, particularly of the distal end of the thick limb of the loop of Henle. The thick limb, identified by antibody to Tamm-Horsfall protein, ended abruptly, either at the macula densa or at a variable distance after it. At this point there was an abrupt change in cell size. Confocal microscopy and other techniques showed that this point marked an abrupt beginning of tubular staining by the cytokeratin antibody PKK2 and the lectin UEA 1, with an abrupt end of staining by the lectin DBA. Distal from this point, there were gradual changes in staining of the tubule by various reagents including other antibodies to cytokeratins. These structural findings suggest that there is a fundamental change in the tubule at the end of the thick limb. The abrupt end to the thick limb in man resembles that seen in the rat and the rabbit.


The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse duodenal epithelium.

RNA synthesis was examined by radioautography in mouse duodenal epithelium using 3H-uridine as a tracer of the salvage pathway and 3H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after 3H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After 3H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The 3H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the 3H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.


Effect of spinal microinjections of an antagonist to substance P or somatostatin on the exercise pressor reflex.

The purpose of this study was to determine the heart rate and arterial blood pressure changes to isometric skeletal muscle contraction and muscle stretch before and after microinjecting an antagonist to substance P (SP) or somatostatin (SOM) into the L-7 dorsal horn region of the spinal cord of anesthetized cats. Anesthesia was induced by administering an anesthetic gas mixture and was subsequently maintained with alpha-chloralose. Triceps surae contraction was induced by electrically stimulating the L-7 ventral root. Three muscle manipulations (all 1 minute in duration) were performed: 1) continuous tetanic contraction, 2) intermittent tetanic contractions (1 second of contraction, 1 second of relaxation), and 3) passive muscle stretch. Saline microinjections had no effect on the cardiovascular responses to these muscle manipulations. However, both peptide antagonists blunted the pressor response to a continuous tetanic contraction as mean arterial pressure increased 47 +/- 4 and 44 +/- 4 mm Hg before and 28 +/- 3 and 28 +/- 4 mm Hg after microinjecting the SP or SOM antagonist, respectively. In contrast, neither antagonist influenced the increase in mean arterial pressure produced by passive stretch; values were 43 +/- 6 versus 41 +/- 6 mm Hg (SP antagonist) and 39 +/- 7 versus 42 +/- 7 mm Hg (SOM antagonist) before and after injections, respectively. Microinjecting the SOM antagonist attenuated the pressor response to intermittent tetanic contractions (44 +/- 4 mm Hg before SOM antagonist versus 26 +/- 4 mm Hg after SOM antagonist), whereas the SP antagonist had no effect (35 +/- 3 mm Hg before SP antagonist versus 32 +/- 4 mm Hg after SP antagonist).(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacokinetics of tissue-type plasminogen activator during acute myocardial infarction in men. Effect of a prostacyclin analogue.

Coronary reocclusion complicates the thrombolytic therapy of acute myocardial infarction despite the routine use of aspirin. This is consistent with experimental studies demonstrating that multiple agonists, in addition to thromboxane A2, mediate the platelet activation underlying reocclusion. Consequently, a more potent antiplatelet therapy with a broader spectrum of activity than aspirin may be required in this setting. Prostacyclin and its more stable analogue, iloprost, inhibit platelet aggregation to all known agonists and exert an additional effect over aspirin alone. Experiments in animal models have demonstrated, however, that iloprost increases the clearance of tissue-type plasminogen activator (t-PA) and impairs thrombolysis in vivo. This study examines whether a similar interaction occurs in humans. Twelve patients with acute myocardial infarction received t-PA intravenously, 60 mg in the first hour and a maintenance infusion of 13.3 mg/hr for 3 hours. Patients were assigned in a double-blind fashion to iloprost (2 ng/kg/min) or placebo following the initial 90 minutes of the maintenance infusion of t-PA. Iloprost decreased mean arterial blood pressure (-10 +/- 2.9 mm Hg, p less than 0.05) but did not alter heart rate. Steady-state plasma iloprost concentration was 591 +/- 64 pmol/l. At this concentration, iloprost markedly inhibited platelet aggregation in vitro, particularly in the presence of aspirin. Steady-state clearance of t-PA was unchanged by iloprost (454 +/- 65 versus 443 +/- 136 ml/min in controls, p = NS). Furthermore, neither elimination kinetics nor plasma protein binding of t-PA was altered by iloprost. At plasma levels that exert a potent antiplatelet effect, iloprost did not alter the pharmacokinetics of t-PA in men. Prostacyclin analogues may prove useful as an adjunct to plasminogen activators, particularly in patients at high risk for thrombotic reocclusion.


Adrenergic effects on the biology of the adult mammalian cardiocyte.

To delineate the mechanism(s) of catecholamine-mediated cardiac toxicity, we exposed cultures of adult cardiac muscle cells, or cardiocytes, to a broad range of norepinephrine concentrations. Norepinephrine stimulation resulted in a concentration-dependent decrease in cardiocyte viability, as demonstrated by a significant decrease in viable rod-shaped cells and a significant release of creatine kinase from cells in norepinephrine-treated cultures. Norepinephrine-mediated cell toxicity was attenuated significantly by beta-adrenoceptor blockade and mimicked by selective stimulation of the beta-adrenoceptor, whereas the effects mediated by the alpha-adrenoceptor were relatively less apparent. When norepinephrine stimulation was examined in terms of cardiocyte anabolic activity, there was a concentration-dependent decrease in the incorporation of [3H]phenylalanine and [3H]uridine into cytoplasmic protein and nuclear RNA, respectively. The decrease in cytoplasmic labeling was largely attenuated by beta-adrenoceptor blockade and mimicked by selective stimulation of the beta-adrenoceptor, but alpha-adrenoceptor stimulation resulted in relatively minor decreases in cytoplasmic labeling. The norepinephrine-induced toxic effect appeared to be the result of cyclic AMP-mediated calcium overload of the cell, as suggested by studies in which pharmacological strategies that increased intracellular cyclic AMP led to decreased cell viability, as well as studies that showed that influx of extracellular calcium through the verapamil-sensitive calcium channel was necessary for the induction of cell lethality. Additional time-course studies showed that norepinephrine caused a rapid, fourfold increase in intracellular cyclic AMP, followed by a 3.2-fold increase in intracellular calcium [( Ca2+]i). These results constitute the initial demonstration at the cellular level that adrenergic stimulation leads to cyclic AMP-mediated calcium overload of the cell, with a resultant decrease in synthetic activity and/or viability.


Substance P-induced granulocyte infiltration in mouse skin: the mast cell-dependent granulocyte infiltration by the N-terminal peptide is enhanced by the activation of vascular endothelial cells by the C-terminal peptide.

Previous studies have shown that substance P induces granulocyte infiltration in mouse skin, which is mediated through mast cell degranulation. However, it is not yet known whether the direct effect of substance P on vascular endothelial cells is involved in the granulocyte infiltration in the skin. To solve this issue, we used the N-terminal peptide substance P1-9 (SP1-9), which is active for mast cells but inactive for vascular endothelial cells, and the C-terminal peptide SP6-11, which is active for vascular endothelial cells but inactive for mast cells, since substance P activates both mast cells and vascular endothelial cells. The subcutaneous administration of substance P (10(-7)-10(-5)M) caused granulocyte (neutrophil and eosinophil) infiltration in the skin of BALB/c mice 6 h after the injection. SP1-9 (10(-5)-10(-4) M) also caused granulocyte infiltration of mouse skin which was associated with mast cell degranulation. In contrast, SP6-11 (10(-7)-10(-4) M), which was found to increase the vascular permeability of endothelial cells in mouse skin, induced no significant granulocyte infiltration nor mast cell degranulation. However, SP6-11 (10(-5)-10(-4) M) enhanced SP1-9-induced granulocyte infiltration in the skin without any significant increase in mast cell degranulation. We conclude that substance P causes granulocyte infiltration in mouse skin through both mast cell degranulation induced by the N-terminal peptide of substance P and the activation of vascular endothelial cells induced by the C-terminal peptide of substance P.


Expression of adhesion molecules in human intestinal graft-versus-host disease.

The distribution of three cellular adhesion molecules, ICAM-1, ELAM-1 and VCAM-1, was studied in normal rectal mucosa and in graft-versus-host disease (GvHD) using immunohistological and morphometric techniques. In normal controls, ICAM-1 was demonstrable on endothelial cells and leucocytes within the lamina propria, ELAM-1 on endothelial cells only and VCAM-1 on lamina propria leucocytes, many of which exhibited long dendritic processes surrounding the glands. In GvHD, the enterocytes became positive for ICAM-1 and this was often associated with the presence of intra-epithelial LFA-1+ lymphocytes and macrophages, the latter containing debris of apoptotic cells. The staining was, however, restricted to the luminal membrane of the epithelial cells, raising doubts about the role of ICAM-1 as a ligand for LFA-1 on mucosal leucocytes in rectal GvHD. ELAM-1 expression was increased in GvHD both in terms of the length of positive endothelium and staining intensity. VACM-1 was increased on endothelial cells but not leucocytes in the lamina propria in contrast to our previous findings in cutaneous GvHD where VCAM-1+ dendritic cells were increased and endothelial cells remained negative. Normal patterns of adhesion molecule staining were seen in two biopsies exhibiting no morphological evidence of GvHD, from patients who had strong clinical evidence of the disease, indicating that immunostaining for these molecules is unlikely to be of help in improving the sensitivity of histological diagnosis. However, the possibility that adhesion molecule staining may be useful in improving diagnostic specificity by helping to distinguish GvHD from identical histological changes produced by irradiation and cytotoxic drugs is worthy of further investigation.


Enhancement of the antigen-presenting function of monocytes by cholesterol: possible relevance to inflammatory mechanisms in extrinsic allergic alveolitis and atherosclerosis.

Extrinsic allergic alveolitis (EAA) (synonym: hypersensitivity pneumonitis) is a hypersensitivity lung disease characterized by lymphocytic infiltrates in the pulmonary interstitial tissues. We have previously reported that the numbers of lymphocytes in bronchoalveolar lavage (BAL) samples in this disease correlate with levels of cholesterol and neutral lipid-laden 'foamy' macrophages. We have also reported that the macrophages express an increased density of MHC class II antigens (in particular HLA-DQ) which are known to be essential for antigen recognition by T lymphocytes. The aim of the present study was to explore whether cholesterol is capable of enhancing the antigen-presenting function of mononuclear phagocytes by modulating the expression of HLA-D region products. Incubation of purified monocytes from healthy volunteers with cholesterol in serum-free medium induced a significant increase in both the percentages of monocytes expressing HLA-DQ (P less than 0.02) and in the intensity of expression of the three HLA-D sub-region products, HLA-DQ, -DP and -DR (P less than 0.02, less than 0.01, less than 0.05, respectively). The cholesterol pre-incubated monocytes also exhibited enhanced antigen-presenting function (P less than 0.05), compared with controls pre-incubated without cholesterol. These findings indicate that increases in cholesterol in the extracellular milieu may augment antigen presentation by modulating the expression of HLA-D region products on antigen-presenting cells. Apart from EAA, this observation may also have relevance to inflammatory mechanisms in atherosclerosis, where 'foamy' macrophages also occur in association with hypercholesterolaemia.


Concomitant augmentation of CD4+ CD29+ helper inducer and diminution of CD4+ CD45RA+ suppressor inducer subset in patients infected with human T cell lymphotropic virus types I or II.

To examine the immunomodulatory effects of HTLV infection, lymphocyte subset analysis was performed on patients infected with human T cell lymphotropic virus type-I (HTLV-I, n = 6) or -II (HTLV-II, n = 12) and on normal blood donors (n = 16). The percentages of total B lymphocytes (CD19), natural killer (NK) cells (CD16), T lymphocytes and their subsets (CD2, CD3, CD4, CD5, CD7, CD8), and IL-2R (CD25) were found to be within the range found in normal donors. However, the expression of CD8+ HLA-DR+ increased significantly in patients with HTLV-I or HTLV-II infection (14.1 +/- 3.9% and 9.7 +/- 2.4% respectively; P less than 0.01) when compared with controls (3.2 +/- 1.1%). In addition, there was a significantly greater proportion of CD4+CD29+ T lymphocytes (29.3 +/- 6.1% and 31.1 +/- 9.0%; P less than 0.05) with concomitant diminution of CD4+CD45RA+ T lymphocytes (8.3 +/- 3.3% and 11.4 +/- 1.5%; P less than 0.01) in patients infected with HTLV-I or HTLV-II respectively, when compared with controls. The increased percentage of CD4+CD29+ subpopulations showed a direct correlation (rs = 0.86; P less than 0.001) with HTLV-specific antibody production. No difference in the CD8 population coexpressing CD29 and S6F1 (an epitope of LFA-1) were observed in the HTLV-infected group when compared with normal donors and functional analysis exhibited minimal cytotoxicity against lectin labelled heterologous target cells. Thus, the shift in the suppressor/cytotoxic to helper/inducer 'memory' CD4+ may be associated with immunoregulatory abnormalities often found in persons infected with HTLV-I or HTLV-II.


HBsAg in urine: a new approach for the detection of urinary antigens.

In order to define the optimal conditions for detection of microbial antigens in urine, urinary HBsAg excreted during hepatitis B was chosen as a model. Using commercial kits, which mainly involve anti-discontinuous epitopes, we found urinary HBsAg in only 50% of patients with HBsAg in their sera. In contrast, with an inhibition method involving a monoclonal antibody recognizing a continuous epitope, urinary HBsAg was found in 100% of these patients. Structural analysis of HBsAg showed that urinary HBsAg is denatured; it can escape detection by commercial kits well fitted for detection of native serum HBsAg. General implications for the revelation of urinary microbial antigens are discussed.


Biochemical and functional characterization of the leucocyte tyrosine phosphatase CD45 (CD45RO, 180 kD) from human neutrophils. In vivo upregulation of CD45RO plasma membrane expression on patients undergoing haemodialysis.

The biochemical and functional characterization, and the regulation of plasma membrane expression of the leucocyte tyrosine phosphatase CD45, have been investigated in neutrophils from healthy donors and patients undergoing haemodialysis. CD45 proteins of 180 kD and 130-150 kD were precipitated from neutrophils from both healthy subjects and haemodialysed patients. Prolonged storing, as well as trypsin treatment of samples containing the 180-kD CD45 protein, generated the 130-150-kD polypeptides. The 130-150-kD CD45 polypeptides carried extracellular CD45 epitopes, including the sialic acid-related UCHL1 epitope (CD45RO). Furthermore, these trypsin-generated CD45 polypeptides did not possess phosphatase activity, which could be detected on the 180-kD protein. A remarkable quantitative increase of cell surface expression of the neutrophil CD45 components was detected both after in vitro neutrophil activation and after dialysis treatment with neutropenic membranes. The CD45 biochemical pattern did not qualitatively change upon either in vitro or in vivo dialysis-induced neutrophil activation. The upregulated expression of CD45 on neutrophils from dialysed patients correlated with the neutropenic effect induced by the different dialyser membranes. Maximal upregulation of CD45 expression was observed after 15 min of dialysis with neutropenic membranes, and normal expression levels were restored after 1 h. By contrast, increase of CD45 plasma membrane expression induced in vitro by treatment of normal neutrophils with the degranulatory agents fMLP or Ca2+ ionophore was maintained. These results demonstrate that neutrophil cell surface expression of the 180-kD CD45 protein is upregulated during the in vivo haemodialysis process, and suggest that a proteolytic activity could regulate the enzymatic activity of CD45 by degranulation of its cytoplasmic phosphatase domains.


Long-lasting tolerance of articular cartilage after experimental intraoperative radiation in rabbits.

In experimental intraoperative irradiation, 18 adult rabbits received a single, 50-Gy dose of x-radiation at a unilateral knee joint, and subsequent changes in the articular cartilage were examined over a 15-month period by histology, scanning electron microscopy, and autoradiography. Although the subchondral bone showed histologically typical findings of osteonecrosis three to nine months postirradiation, the articular cartilage revealed no obvious degenerative changes during the entire study period. Scanning electron microscopy revealed normal collagen architecture in the irradiated cartilage for as long as 15 months postirradiation. Autoradiography demonstrated active RNA synthesis by the irradiated chondrocytes during the same period. These results indicate that articular cartilage tissue tolerates intraoperative radiotherapy without sustaining serious degenerative changes, unless possible collapse or contracture disturbs its biomechanical integrity. The survival of articular cartilage can be advantageous for this type of limb-salvage surgery in the treatment of malignant bone tumors around a synovial joint.


Evidence for a role of free radicals by synthesized scavenger, 2-octadecylascorbic acid, in cerulein-induced mouse acute pancreatitis.

To define the role of free radicals and of lipid peroxide involvement during the progress of cerulein-induced acute pancreatitis in mice, we evaluated the effect of a novel free radical scavenger, 2-octadecylascorbic acid (CV-3611), on pancreatic edema formation, and the levels of serum enzymes (amylase, lipase) and of lipid peroxide in pancreatic tissue. Mice were divided into three groups: control group, intraperitoneal injection of saline only; pancreatitis group, cerulein 50 micrograms/kg injected intraperitoneally six times at 1-hr intervals; treatment groups, CV-3611 10 mg/kg subcutaneously just after intraperitoneal cerulein injection. After the cerulein injection, the degree of pancreatic edema formation, serum amylase and lipase levels, and the amount of lipid peroxide in pancreatic tissue increased significantly during the observation period of 12 hr. Treatment with CV-3611 resulted in significant reduction in pancreatic edema formation at 3.5 hr (P less than 0.05) and 9 hr (P less than 0.05), serum amylase and lipase levels at 3.5 hr (P less than 0.05) and 12 hr (P less than 0.05), and lipid peroxide levels at 3.5 hr (P less than 0.05), 6 hr (P less than 0.05) and 12 hr (P less than 0.05). These results indicate that a novel free radical scavenger, CV-3611, has a strong therapeutic effect during the development of acute pancreatitis and suggest that oxygen-derived free radicals play an important role in the pathogenesis of acute pancreatitis.


Cerulein-induced acute pancreatitis in the rat. Study of pancreatic secretion and plasma VIP and secretin levels.

A study was made with different doses of cerulein (2, 4, 10 and 20 micrograms/kg) administered subcutaneously to rats by four injections at intervals of 1 hr; the aim of this work was to study exocrine pancreatic secretion of the rat under cerulein-induced acute pancreatitis, analyzing enzyme and hydroelectrolyte secretion of pancreatic juice. A further aim was to study the relationship between the dose of cerulein and the plasma levels of peptides controlling hydroelectrolyte secretion of the pancreas, like secretin and vasoactive intestinal peptide (VIP). At the lowest dose schedule, the amounts of total protein and enzymes (amylase and trypsin) in pancreatic juice decreased significantly, plasma amylase increased, and the pancreas became edematous. Higher doses magnified these effects. By contrast, ductular function (flow and HCO3-) was well preserved in cerulein-treated rats, and this was probably due to the significant increase in plasma levels of immunoreactive secretin whereas VIP levels were unchanged. The secretin released by treatment with cerulein is able to palliate the lack of flow from acinar origin that is affected in the process of acute pancreatitis, being a beneficial response to the cerulein treatment.


Isolation and characterization of a novel glycosyl-phosphatidylinositol-anchored glycoconjugate expressed by developing neurons.

In search of new markers for studying thymic and nervous system ontogeny, we raised rat monoclonal antibodies against glycosyl-phosphatidylinositol-anchored molecules among which larger groupings have been shown to be ectoenzymes and adhesion molecules. Two of these monoclonal antibodies (H193-4 and H194-563, IgG) were found to recognize glycosyl-phosphatidylinositol-anchored glycoconjugates of 28-33 kDa (P31) and 50-70 kDa in developing mouse brain and thymus respectively, when these tissues were analysed by immunoblot experiments. P31 antigen was found to be transiently expressed by neurons in neural primary cultures [Rougon, G., Alterman, L., Dennis, K., Guo, X. J. & Kinnon, K. (1991) Eur. J. Immunol. 21, 1397-1402]. We show in this report that, in developing mouse brain, a maximal expression occurred between embryonic day 17 and post-natal day 5, a period that corresponds to the formation of neuronal networks. P31 antigen was immunopurified and found to possess the following properties: (a) it was soluble in alkaline solvents; (b) it bound to DEAE-cellulose and was eluted by a salt gradient of 0-1 M NaCl; (c) it was sensitive to endoglycosidase F digestion; (d) it was insensitive to heparinase, hyaluronidase, chondroitinase ABC, endo-beta-galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoreactivity; (f) it contained phosphate; (g) it lost its immunoreactivity after treatment with phosphatidylinositol phospholipase C and treatment. These characteristics combine to suggest that P31 is an anionic glycoconjugate sharing similarities with Leishmania donovani lipophosphoglycan and with the heat-stable antigen recognized by J11d antibody on murine hematopoïetic cells. This last hypothesis was further confirmed by the observation that oligonucleotide probes derived from the heat-stable antigen-encoding cDNA detect, in developing brain, a 1.8-kb mRNA species similar in size to that reported for the heat-stable antigen mRNA and following the same developmental expression as P31 antigen.


Estrogen induction of alcohol dehydrogenase in the uropygial gland of mallard ducks.

Treatment of mallard ducks with estradiol, or a combination of estradiol and thyroxine, has been shown to result in the proliferation of peroxisomes and production of diesters of 3-hydroxy fatty acids, the female pheromones, in the uropygial gland of male and female mallard ducks. Such a treatment results in the induction of a unique set of proteins. A cDNA library enriched in hormone-induced transcripts was subjected to differential screening. The nucleotide sequence of one of the two unique cDNA clones, DGH1, had high similarity to the Human class I alcohol dehydrogenase (ADH) gamma subunit and represented the carboxy-terminus of the protein from amino acid 190-374. SDS/PAGE and Western blot analysis of the proteins indicated that the level of a 38-kDa protein that cross-reacted with antibodies prepared against the chicken ADH was increased 5-7-fold by hormone treatment. Assays for ADH activity in the uropygial gland extracts of male mallards showed a 5-7-fold induction of the enzyme by hormone treatment. The 1.9-kb ADH mRNA levels were increased 12-14-fold under these conditions. Of all the tissues tested, the uropygial gland had the highest levels of ADH mRNA. Induction of ADH by estradiol treatment occurred only in this tissue. Elevated levels of ADH were also observed in the glands of male mallards in eclipse, the post-nuptial condition when the hormonal balance is shifted to higher estrogen levels, suggesting that this enzyme is regulated by estrogens in this period. Estradiol treatment caused an 80% decrease in the NAD+/NADH ratio in the uropygial gland and a twofold increase in the fatty alcohol oxidation rate catalyzed by the gland extract. These observations could help explain how increased levels of ADH could contribute to the production of the diesters.


Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.


Physarum vitronectin-like protein: an Arg-Gly-Asp-dependent cell-spreading protein with a distinct NH2-terminal sequence.

A 70-kDa protein cross-reacted with anti-bovine vitronectin was isolated from slime mold Physarum polycephalum. The NH2-terminal amino acid sequence of the protein, referred to as Physarum vitronectin-like protein, did not share any homology with those of animal vitronectins. It had cell-spreading activity, which was specifically inhibited by an Arg-Gly-Asp (RGD)-containing peptide.


Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel.

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.


Positively reinforcing effects of the neurokinin substance P in the basal forebrain: mediation by its C-terminal sequence.

The conditioned corral preference paradigm was used to assess reinforcing effects of substance P (SP) and its N- and C-terminal fragments injected unilaterally into the region of the nucleus basalis magnocellularis (NBM) in rats. Behavioral testing was carried out in a circular open field, consisting of 4 quadrants equally preferred by the animals prior to conditioning. A single conditioning trial was performed. Rats received one microinjection (0.5 microliter) of SP (0.74 pmol), of the N-terminal fragment SP (1-7) and the C-terminal fragment analog DiMe-C7 (each at doses of 0.074, 0.74, and 74 pmol), or vehicle (phosphate-buffered saline; PBS). After injection the rats were placed into the open field with the four quadrants being separated by Plexiglas barriers (closed corral). During the test for conditioned corral preference, when provided a choice between the four quadrants, only those rats injected with SP and the equimolar dose of DiMe-C7 (0.74 pmol) spent more time in the treatment corral, indicative of a positively reinforcing action. None of the other doses of DiMe-C7 and of SP(1-7) influenced the preference behavior. For rats injected with 0.74 pmol SP, SP (1-7), and DiMe-C7, a behavioral analysis was performed for the 15 min conditioning trial. SP and DiMe-C7 reduced rearing and grooming behavior, whereas DiMe-C7 and SP(1-7) increased locomotor activity. However, the acute behavioral effects of SP and its fragments were not correlated with the subsequent place preference behavior during the test trial. The results are discussed in the framework of a structure/activity relationship for the positively reinforcing properties of SP in the region of the NBM. Furthermore, neuropathological implications of the present data are considered, since the homologous nucleus basalis of Meynert in man is known to degenerate in Alzheimer's disease, which is characterized behaviorally by a progressive deterioration in associative functioning.


Effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa.

To study by a triple stain technique the effect of sperm-immobilizing antibodies on the acrosome reaction of human spermatozoa. The spermatozoa were allowed to swim up and culture in a medium containing 7.5% (vol/vol) serum with sperm-immobilizing antibodies or control serum up to 6 hours. Sperm mobility was analyzed, and the percentage of live acrosome reacted spermatozoa was determined. Samples were collected from patients referred to university hospital infertility clinics. Serum samples were drawn from seven patients with sperm-immobilizing antibodies. All the sera were heat activated and stored at -40 degrees C until use. Semen samples were taken from two healthy donors. During culture for 6 hours, the percentage of live sperm showing the acrosome reaction increased significantly (P less than 0.01) in the control group but not in the sperm-immobilizing antibodies group. However, the inhibitory effect of sperm-immobilizing antibodies on the acrosome reaction was reversed when sperm was reincubated in medium with control serum (P less than 0.01). Sperm-immobilizing antibodies block fertilization at least in part by inhibiting the acrosome reaction of human spermatozoa.


Identification of insulin-like growth factor binding proteins in human oviduct.

To determine if human oviduct expresses messenger ribonucleic acids (mRNAs) encoding insulin-like growth factor binding proteins (IGFBPs), if oviductal epithelium secretes IGFBPs into conditioned medium (CM), and if IGFBP secretion is regulated by steroid hormones. Northern blots of RNA, isolated from late proliferative phase human fimbria and oviductal isthmus, were probed with complementary deoxyribonucleic acids encoding IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. In addition, oviductal ampullary epithelial cells were cultured with and without estrogen and/or progesterone (P), and IGFBPs were examined in CM by Western ligand blot analysis and identified using specific antisera. Tissue was obtained from hysterectomy specimens at Stanford University Hospital, a private teaching institution. Patients undergoing hysterectomy for benign disease. None. Transcripts of IGFBP mRNA and IGFBPs secreted into CM were detected by autoradiography of Northern and Western ligand blots, respectively. Messenger RNA transcripts encoding IGFBP-2, -3, and -4 were detected, whereas IGFBP-1 mRNA was barely detectable in oviductal tissue. In CM, IGFBP-2 and IGFBP-3 were detected, as was a unique 24-kd IGFBP, although IGFBP-1 was not observed. Estrogen and/or P did not regulate the secretion of these IGFBPs by cultured oviductal epithelium. Human oviduct expresses mRNAs encoding IGFBP-2, IGFBP-3, and IGFBP-4, and in vitro oviductal epithelium secretes IGFBP-2, IGFBP-3, and a unique binding protein of 24 kd, which may be the recently identified IGFBP-4.


Incidence of sperm with two fluorescent bodies in men with impaired fertility.

To determine the two fluorescent body (2FLB) frequency in males with impaired fertility. Males of couples were evaluated along with their female partners for reproductive function. They were classified as having the potential for fertility (control group) or as infertile (test group). This was based on the evaluation of both members of the couple. Semen parameters were determined and analyzed for differences among diagnostic groups. The setting was clinical secondary care in both private and institutional practice. The study population was composed of males of couples who were being evaluated for infertility. Classification as to reproductive potential and categories of abnormality was based on measurement of semen parameters and clinical judgment. Semen specimens were obtained and analyzed from 78 control and 93 test subjects. Semen parameters including volume, sperm count, sperm motility, percent morphologically abnormal sperm, and percent of quinacrine-stained sperm containing zero, one, or two FLBs were determined. The test group was found to have a 2FLB frequency that was significantly different from that of the control group. The data were then analyzed for significant differences in 2FLB frequencies in subpopulations. When subjects from all diagnostic subgroups that included varicocele were compared with the controls, the 2FLB frequency was significantly elevated (median percent of 2FLB was 1.2 versus 0.7). The 2FLB frequency in the remaining diagnostic groups was not elevated. This is the first report of an elevated 2FLB frequency associated with a biological abnormality. The cause of the elevated 2FLB frequency is not known. However, other anomalies that have been identified in varicocele patients have been associated with elevated intrascrotal temperatures. There are data available that associates elevated temperature with aneuploidy in mouse oocytes.


Alpha 2-macroglobulin and alpha 2-macroglobulin/proteinase complexes in human seminal fluid.

To determine the broad-spectrum proteinase inhibitor alpha 2-macroglobulin (alpha 2M) and its functional subforms, i.e., free alpha 2M and alpha 2M/proteinase complexes, in human seminal fluid by using specific enzyme immunoassay systems. The study has been performed in the Andrology Department of the Dermatology Clinics and the Laboratory of Immunopathology of the Institute of Immunology. PATIENTS, PARTICIPATES: Routine patients attending the Andrology Department. The data have been obtained without particular interventions before or after collection of seminal fluid. The aim of the study was to determine whether alpha 2M or alpha 2M/proteinase complexes are present in human seminal fluid. The concentration of total alpha 2M in human seminal fluid ranged from 1 to greater than 1,000 ng/mL, and between 56% and 85% of the inhibitor was complexed with proteinases. These findings show that alpha 2M and alpha 2M/proteinase complexes have to be considered as functionally relevant biomolecules in male genital tract secretions.


Seroprevalence of hepatitis C virus nucleocapsid antibodies in patients with cryptogenic chronic liver disease.

The serological responses to two different hepatitis C virus antigens were studied by enzyme-linked immunosorbent assay in a variety of chronic liver diseases and in healthy blood donors. The study population comprised 97 cases of cryptogenic chronic liver disease (40% with a history suggestive of parenterally transmitted non-A, non-B hepatitis and 60% without such a history), 87 cases of other well-characterized chronic liver diseases and 96 voluntary blood donors. The commercially available C100-3 assay and a new assay utilizing a 22 kD recombinant protein (c22) from the nucleocapsid region of the virus were used for antibody detection. Overall in the non-A, non-B hepatitis group, 77% were positive for anti-c22, 55% were positive for anti-C100-3 and 24% were negative by both tests. In the parenterally transmitted chronic liver disease group, 82% were positive for anti-C100-3 and 90% were positive for anti-c22 (not significant). In the cryptogenic chronic liver disease cases 36% were positive for anti-C100-3 and 67% were positive for anti-c22 (p less than 0.001). Only in one case (a patient with hepatitis B virus infection) was anti-C100-3 detected without concomitant anti-c22. None of the voluntary blood donors had detectable hepatitis C virus antibodies. The new enzyme-linked immunosorbent assay test for anti-c22 would appear to be a more sensitive indicator of chronic hepatitis C virus infection than the existing commercial test, suggesting a useful diagnostic role in both cases of cryptogenic chronic non-A, non-B hepatitis liver disease and for the screening of blood products for prevention of hepatitis after transfusion.


Antibodies against synthetic oligopeptides deduced from the putative core gene for the diagnosis of hepatitis virus infection.

Immunoassays were developed to detect antibodies against oligopeptides deduced from the putative core gene of hepatitis C virus, and their performances were compared with that of the commercial immunoassay for antibodies against the product of nonstructural regions of hepatitis C virus (anti-C100-3). A 19-mer oligopeptide (CP10) and a 36-mer oligopeptide (CP9) were chemically synthesized, which represented hydrophilic regions of the product of the hepatitis C virus core gene. They were used to capture corresponding antibodies, anti-CP10 and anti-CP9, by enzyme-linked immunosorbent assay in sera from patients with acute or chronic non-A, non-B liver disease and in blood donations. At the onset of acute non-A, non-B hepatitis, anti-CP10 was detected in 15 of 20 patients (75%), and anti-CP9 was detected in 14 patients (70%). This was more frequent than anti-C100-3, which was found in only 9 patients (45%). In 186 patients with chronic non-A, non-B liver disease, anti-CP9, anti-CP10 or both were detected in 170 patients (91%). This was more frequent than anti-C100-3, which was found in 138 patients (74%). Blood with anti-CP10 as the single serological marker for hepatitis C virus infection transmitted non-A, non-B hepatitis by needlestick exposure. In sera from 558 apparently healthy blood donors, anti-CP10 was detected in 55 donors (9.9%), anti-CP9 was detected in 26 donors (4.7%) and anti-C100-3 was detected in 7 donors (1.3%).(ABSTRACT TRUNCATED AT 250 WORDS)


Vascular adhesion molecules in acute and chronic liver inflammation.

Adhesion to and penetration through the sinusoidal vascular endothelium is a mandatory step for leukocyte migration and accumulation at sites of liver inflammation. This leukocyte trafficking is controlled by interactions between adhesion molecules on leukocytes and corresponding ligands on endothelial cells. We have analyzed the in situ distribution of two recently described vascular adhesion molecules (i.e., endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1) and of the lymphocyte "homing" receptor cluster of differentiation antigen-44 in normal and inflamed liver biopsy specimens. Endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 were absent from normal liver tissue, but they were strongly expressed on sinusoidal lining cells in inflammatory liver disease. Endothelial leukocyte adhesion molecule-1 expression predominated diffusely throughout the liver parenchyma in acute hepatitis; in contrast, vascular cell adhesion molecule-1 was mainly expressed in areas of periportal and intralobular inflammation in chronic active and persistent hepatitis. The "homing" receptor cluster of differentiation antigen-44 was weakly expressed on scattered mononuclear cells and on sinusoidal lining cells in normal liver tissue, but it was strongly up-regulated on mononuclear inflammatory cells and sinusoidal lining cells in acute and chronic hepatitis. In addition, reactivity for the cluster of differentiation antigen-44 was found on the membranes of variously sized clusters of hepatocytes in biopsy specimens with acute hepatitis. De novo or up-regulated expression of these adhesion molecules on sinusoidal lining cells in inflamed liver biopsy specimens indicates that these cells actively modulate their phenotype in response to environmental factors, thus playing a key role in the recruitment of leukocytes in acute and chronic liver inflammation.


Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes.

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into plasma cells. CD19 is a member of the Ig gene superfamily with two extracellular Ig-like domains separated by a non-Ig-like domain, and with an extensive approximately 240 amino acid cytoplasmic domain. In this study, Southern blot analysis revealed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned approximately 8 kilobases (kb) of DNA in human and approximately 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5' of the putative translation initiation AUG codon was well conserved in sequence between human and mouse and contained potential transcription regulatory elements. In addition, the 3' untranslated regions (UT) of the CD19 genes following the termination codon were conserved in sequence. The high level of conservation of nucleotide sequences between species in all exons and 5' and 3' UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.


Characterization of epithelial phenotypes in mortal and immortal human breast cells.

We have previously described the mortal human breast epithelial culture MCF-10M, that was derived from fibrocystic breast tissue, was cultivated in medium with low calcium content for over 2 years, and spontaneously gave rise to the immortal MCF-10 cell line. The emergence of immortalized cells, characterized by growth in conventional calcium levels, from mortal cells has proven to be a reproducible event. Here we report the establishment of a second immortal line from MCF-10M, designated MCF-10-2, and establishment of the MCF-12 immortal line after long-term cultivation of MCF-12M mortal cells from reduction mammoplasty tissue. DNA fingerprinting demonstrated the independent, human origin and lineage of the MCF-10-2 and MCF-12 cell lines. Both lines require cortisol and EGF for maximal growth. The expression in these cultures of in vivo breast epithelial phenotypes was analyzed using 2-dimensional gel Western blots and immunoperoxidase staining with antibodies to cytokeratins and polymorphic epithelial mucin. MCF-10M and MCF-12M retain the cytokeratin profile of the luminal cell (7, 8, 18, 19), and also express cytokeratin 14, found predominantly in basal cells. The immortal lines express a similar profile, except that cytokeratin 19, a component of the fully differentiated luminal cell, is not expressed in the more uniform population seen in MCF-10 and MCF-12, but is retained in the morphologically mixed, less-selected population of MCF-10-2. Epitopes on the polymorphic epithelial mucin, recognized by antibodies HMFG 1, HMFG 2 and SM-3, were detected in the mortal cultures and in the immortal lines, indicating the occurrence of both normal and abnormal mucin processing. MCF-10, MCF-10-2 and MCF-12 cells do not form tumors in nude mice, but appear to organize as duct-like structures before regressing in the 5th week post injection.


Cardiovascular responses of patients with cardiac disease to talking and exercise stress testing.

The blood pressure (BP), heart rate (HR), and premature ventricular contraction responses of patients with cardiac disease during talking were compared with those during exercise stress testing (EST). BPs, HR, and heart rhythm were recorded in 52 patients with cardiac disease before and during talking about an intentionally neutral topic and compared with changes during EST. The systolic and diastolic BP and HR increased significantly during talking. Diastolic BP increased significantly more during talking than during EST stages 1 and 2. The increase in diastolic BP during EST stage 3 did not differ from the increase during talking. HR and systolic BP increased more during EST than during talking. Nine patients had premature ventricular contractions during both EST and talking, two during EST only, and one while talking only. The verbalization protocol provides information about changes in diastolic BP that are not seen with EST. It also may be of value for evaluating heart rhythm, HR, and systolic BP changes for patients whose disabilities preclude EST.


Serological diversity and chemical structures of Campylobacter jejuni low-molecular-weight lipopolysaccharides.

Low-Mr lipopolysaccharides (LPS) of Campylobacter jejuni reference strains for serotypes O:1, O:4, O:23, and O:36 were examined through the liberation of core oligosaccharides by mild acid cleavage of the ketosidic linkage of 3-deoxy-D-manno-2-octulosonic acid residues to the lipid A moiety. The liberated oligosaccharides were examined for chemical structure by compositional analysis and methylated linkage analysis in conjunction with fast atom bombardment-mass spectrometry of permethylated oligosaccharide derivatives. The results showed (i) that the LPS contained short oligosaccharide chains of branched nonrepetitive structure, to many of which N-acetylneuraminic acid residues remained attached by 2----3 linkages to 4-linked D-galactose residues in the core structure; (ii) that serotypical differences, which are not readily defined through qualitatively similar compositions, are clearly reflected in variations in linkage types and sequences of sugar residues in the outer core attached to an inner region of invariable structure; but (iii) that the presence or absence of NeuAc residues does not appear to be a basis for serotypical differences. The results also showed that oligosaccharide chains from LPS of serotypes O:1 and O:4 are distinctly different and are distinct again from those of the cross-reacting serotypes O:23 and O:36, between whose core oligosaccharide chains no differences were found. It is concluded that the structurally variable low-Mr LPS from C. jejuni show greater similarities to the lipooligosaccharides from Neisseria spp. than to the highly conserved core regions of Salmonella species. Those strains (serotypes O:23 and O:36) which also furnish high-Mr LPS are unique among gram-negative bacteria in possessing both low-Mr molecules of the Neisseria lipooligosaccharide type and high-Mr LPS of the Salmonella smooth type.


The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins.

Eight monoclonal antibodies (MAbs) directed against the 14,000-molecular-weight (14K) antigen of Mycobacterium tuberculosis reacted specifically with mycobacteria of the M. tuberculosis complex. The nucleotide sequence of the gene encoding the 14K antigen was determined by using recombinant DNA clones isolated from lambda gt11 and cosmid libraries of the M. tuberculosis genome. The DNA sequence of the 14K protein gene coded for a polypeptide of 144 amino acids with a calculated molecular mass of 16,277 Da. The 14K antigen has a marked homology with proteins belonging to the alpha-crystallin family of low-molecular-weight heat shock proteins, which includes the 18K antigen of M. leprae. The eight MAbs recognized at least four distinct epitopes localized within the following three regions of the 14K protein: amino acids 10 to 92 (MAbs F67-8 and F67-16), amino acids 41 to 92 (F159-1 and F159-11), and amino acids 41 to 144 (F23-41, F24-2, F23-49, and TB68).


Amino acid substitutions in naturally occurring variants of ail result in altered invasion activity.

Yersinia enterocolitica is the causative agent of a variety of gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis. In addition, systemic infections resulting in high mortality rates can occur in elderly and immunocompromised patients. More than 50 serotypes of Y. enterocolitica have been identified, but only a few of them commonly cause disease in otherwise healthy hosts. Those serotypes that cause disease have been divided into two groups, American and non-American, based on their geographical distributions, biotypes, and pathogenicity. We have been studying two genes, inv and ail, from Y. enterocolitica that confer in tissue culture assays an invasive phenotype that strongly correlates with virulence. Some differences between the American and non-American serotypes at the ail locus were noted previously and have been investigated further in this report. The ail locus was cloned from seven Y. enterocolitica strains (seven different serotypes). Although the different clones produced similar amounts of Ail, the product of the ail gene from non-American serotypes (AilNA) was less able to promote invasion by Escherichia coli than was the product of the ail gene from American serotypes (AilA). This difference is probably due to one or more of the eight amino acid changes found in the derived amino acid sequence for the mature form of AilNA compared with that of AilA. Seven of these changes are predicted to be in cell surface domains of the protein (a model for the proposed folding of Ail within the outer membrane is presented). These results are discussed in relation to the growing family of outer membrane proteins, which includes Lom from bacteriophage lambda, PagC from salmonella typhimurium, and OmpX from Enterobacter cloacae.


Membrane-derived oligosaccharides affect porin osmoregulation only in media of low ionic strength.

Gram-negative bacteria grown under conditions of low osmolarity accumulate significant amounts of periplasmic glucans, membrane-derived oligosaccharides (MDO) in Escherichia coli and cyclic glucans in members of the family Rhizobiaceae. It was reported previously (W. Fiedlder and H. Rotering, J. Biol. Chem. 263:14684-14689, 1988) that mdoA mutants unable to synthesize MDO show a number of altered phenotypes, among them a decreased expression of OmpF and an increased expression of OmpC, when grown in a Bacto Peptone medium of low osmolarity and low ionic strength. Although we confirm the findings of Fiedler and Rotering, we find that the regulation of OmpF and OmpC expression in mdoA mutants is normal in cells grown on other low-osmolarity media, eliminating the possibility that MDO itself might control porin expression. Our data suggest that a certain minimal ionic strength in the periplasm is needed for normal porin regulation. In media containing very low levels of salt, this may be contributed by anionic MDO.


Osteofibrous dysplasia of long bones--a reactive process to adamantinomatous tissue.

The most controversial aspect of osteofibrous dysplasia (OFD) is its possible histogenetic relationship to adamantinoma of long bone. Evidence is recently beginning to accumulate that OFD may be a reactive process to regressive adamantinoma. To verify the concept, 13 lesions of OFD were studied again by immunohistochemistry for cytokeratins of different molecular masses, as well as by conventional stainings. In addition, 2 adamantinomas and 6 fibrous dysplasias of the tibia were studied for reference. A small number of spindle- or ovoid-shaped cells scattered individually in the fibro-osseous stroma showed positive reactions for cytokeratins of 55-57 kDa in 2 lesions, and for those of 45-56.5 kDa in 8 lesions of 13 OFDs, although no definite epithelial island could be detected even by immunohistochemistry. Adamantinomas also showed single cytokeratin-positive cells dispersed in fibroblastic stroma, in addition to epithelial islands positive for cytokeratins of both 55-57 kDa and 45-56.5 kDa. All cases of fibrous dysplasia were negative for cytokeratins. During the observation, no case of OFDs progressed to classic adamantinoma. The present study, demonstrating the existence of an intermediate stage between "differentiated adamantinoma" and total elimination of adamantinomatous components, gives further support for the concept that OFD is a secondary reactive process to adamantinomatous tissue. In practice, the existence of single scattered cytokeratin-immunoreactive cells in otherwise typical OFDs may not indicate the truly malignant behaviour of classic adamantinoma, unless discrete epithelioid cell nests are also found.


Epitope mapping of monoclonal antibodies to Torpedo acetylcholine receptor gamma subunits, which specifically recognize the epsilon subunit of mammalian muscle acetylcholine receptor.

Epitopes for four monoclonal antibodies (mAbs) to the gamma subunit of Torpedo nicotinic acetylcholine receptor (AChR), and one mAb crossreactive with the gamma and delta subunits of Torpedo AChR were mapped using overlapping synthetic peptides corresponding to the complete amino acid sequence of Torpedo gamma subunit. The epitopes for all mAbs were within a 50 residue sequence region, on the cytoplasmic surface of the AChR. Three mAbs crossreacted with mammalian muscle AChRs. Two of them specifically recognized the epsilon subunit of AChRs at adult neuromuscular junction. The epsilon-specific mAbs were used, in conjunction with mAbs specific for the alpha and beta subunits and anti-peptide antisera specific for the epsilon, gamma and delta subunits, to identify in Western blots the subunit complement of embryonic and adult bovine muscle AChRs.


DNA sequence analysis and comparison of the variable heavy and light chain regions of two IgM, monoclonal, anti-myelin associated glycoprotein antibodies.

The complete variable heavy and light chain gene sequences of two monoclonal, IgM, anti-myelin associated glycoprotein (MAG) antibodies associated with peripheral neuropathy, are presented. Comparative analysis of the two VH regions has revealed that they are 88% homologous to one another and are both members of the VH3 gene family. They are also highly homologous to a gene which is frequently utilized in the fetal B-cell repertoire. The V kappa light chain gene of one of the antibodies is 99% homologous to a V kappa II gene and the V lambda light chain gene of the other antibody is only 72% homologous to other known V lambda genes. Further analysis of V genes utilized by anti-MAG antibodies should reveal the structural basis for their binding activity.


Tenascin expression in human glioma cell lines and normal tissues.

Tenascin expression was evaluated in 21 human glioma cell lines and in normal adult tissue extracts by Western and Northern blotting. The cell lines differed in their relative expression of tenascin in the cell-associated and supernatant compartments. Glioma cell line tenascin production was not uniformly stimulated by changes in fetal bovine serum concentration in the growth media. In most glioma cell lines and normal tissue extracts, reducing Western blots and Northern blots revealed two tenascin species, respectively: a major 340 kDa polypeptide and a 9 kb RNA transcript accompanied by a less intense 250 kDa polypeptide and 7 kDa RNA species. In U-87 MG and in normal adult kidney extracts, however, the 250 kDa band and 7 kb transcript were more prominent. Quantitation of tenascin in the glioma lines revealed variable levels that were significantly higher than those in the tissue extracts.


Production and characterisation of monoclonal antibodies to the mid-region 37-67 sequence of parathyroid hormone-related protein.

The production and characterisation of monoclonal antibodies (MAb) to the mid-region sequence 37-67 of human parathyroid hormone-related protein (PTHRP) is described. In spite of the poor immunogenicity of this sub-fragment of PTHRP, a high percentage of specific hybrids were produced by boosting with conjugate and free peptide prior to cell fusion. Seven of the MAbs produced cross-reacted with PTHRP37-67, PTHRP1-86 and native forms of PTHRP. Inhibition studies with peptide sub-fragments of PTHRP37-67 indicated that the majority recognised the 45-59 region. In a RIA for PTHRP1-86, detection limits ranged from 0.17 to 0.9 ng PTHRP1-86/tube, and no cross-reaction was found with PTH1-84. Two MAbs 1D11 and 4B10 were shown to be of potential use in measuring PTHRP1-86 in a two-site immunoradiometric assay in combination with either a solid phase consisting of a MAb to PTHRP1-34, or iodinated affinity purified rabbit antibodies to PTHRP1-34. MAb 1D11 coupled to Sepharose was suitable for immunoextraction of PTHRP, and successfully localised PTHRP on immunoblots. Two additional MAbs were produced which recognised an epitope unique to PTHRP37-67 located in the 37-46 region of the peptide.


Analysis of amplified T cell receptor V beta transcripts by a non-isotopic immunoassay.

We have recently described a new colorimetric DNA enzyme immunoassay (DEIA) for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single stranded DNA and therefore reveals the hybridization event independently from the DNA sequences. Most importantly, the test has an ELISA format and is very rapid and convenient for processing large numbers of samples. In the present report we have adapted this method to reveal the specificity of amplified T cell receptor V beta transcripts. V beta genes were amplified by polymerase chain reaction, using family specific primers and the specificity of the amplified products was determined by Southern blot and by DEIA. Our data demonstrate that DEIA had the same degree of sensitivity and specificity of conventional Southern hybridization. The possibility of analyzing amplified products with the simplicity of a conventional immunoassay should greatly facilitate the analysis of complex multigenic systems such as the T cell receptor and the immunoglobulin repertoire.


Atrial parasystolic trigeminy with sinus node echoes during wandering sinus node pacemaker in a patient with malignant lymphoma and hypertrophic cardiomyopathy.

A case of atrial parasystolic trigeminy associated with shifting or wandering pacemaker in a patient with malignant lymphoma and hypertrophic cardiomyopathy is presented, in which concomitant unsustained sinus node echoes during flattening of sinus P wave were observed. This is the first report to prove that wandering sinus pacemaker was one of the causes of sinus echoes.


Pancreatitis in pediatric human immunodeficiency virus infection.

Because pancreatitis has been reported frequently in adults with human immunodeficiency virus infection, we sought to determine the incidence of pancreatitis in children with acquired immunodeficiency syndrome by reviewing all records of children with AIDS, their serum amylase and lipase levels, and the factors associated with pancreatitis through a case-control analysis. During a 6-year period pancreatitis developed in 9 (17%) of 53 pediatric patients with AIDS. Six children had vertical transmission of infection and three patients had acquired HIV infection through contaminated blood products. Pancreatitis developed at a median age of 5.2 years (range 1.2 to 20 years). All patients had vomiting and abdominal pain. When the patients were first seen, lipase values were elevated more than amylase values (p = 0.028). Amylase and lipase levels declined at comparable rates. In the case-control analysis, pentamidine isethionate was significantly associated with pancreatitis (p = 0.02); the risk was greater in patients who received pentamidine isethionate and had absolute CD4 T-lymphocyte counts less than 100 cells/mm3 (p = 0.001). Infections associated with the onset of pancreatitis included cytomegalovirus (4), Cryptosporidium (1), Pneumocystis carinii pneumonia (3), and Mycobacterium avium intracellulare (1). Coinfection with cytomegalovirus was associated with a protracted course in four children. Ultrasonographic examination demonstrated biliary ductal dilatation 6 months after the onset of pancreatitis in one child. Seven children have died at a mean of 8 months after the initial onset of pancreatitis; the one living child has survived 5 months from the onset of pancreatitis. We conclude that pancreatitis is common in pediatric patients with AIDS and may be related to pentamidine isethionate exposure, especially when absolute CD4 T-lymphocyte counts are less than 100 cells/mm3. Serum amylase levels do not always accurately predict the onset of pancreatitis; serum lipase levels should be measured in children with symptoms. The onset of pancreatitis in an HIV-infected child is a poor prognostic indicator.


Problems of comorbidity in mortality after prostatectomy.

In recent studies of patients with benign prostatic hyperplasia (BPH), men undergoing transurethral resection of the prostate (TURP) had higher long-term mortality than men undergoing open prostatectomy. We tested the hypothesis that the higher mortality for patients undergoing TURP could have occurred if these patients were older and sicker at the time of surgery than patients undergoing open prostatectomy. Retrospective cohort study at Yale-New Haven (Conn) Hospital. Two hundred fifty-two men who underwent TURP or open prostatectomy from 1979 through 1981 for the treatment of BPH. Five-year mortality adjusted for age and severity of comorbid illness at the time of surgery. The crude 5-year mortality rates were 17.5% (22 of 126 patients) for the TURP group and 13.5% (17 of 126 patients) for the open group. At the time of surgery, however, patients in the TURP group were sicker and older than patients in the open group. As the detail and quality of the assessment of comorbidity increased, the adjusted risk of TURP decreased. Improved classifications of comorbidity in three different forms of statistical analysis did not show an effect of type of prostatectomy on long-term mortality (Mantel-Haenszel relative risk, 1.03; 95% confidence interval, 0.57 to 1.87). These results suggest that TURP does not increase long-term mortality after surgery for the treatment of BPH. Inadequate accounting for severity of illness may also affect other statistical "adjustments" used in research concerned with patient outcomes.


Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium.


Common epitopes of human and murine Mallory bodies and Lewy bodies as revealed by a neurofilament antibody.

The antibody SMI 31, which is directed against a phosphorylated epitope, associated with neurofilaments and recognizes Lewy bodies in brains of patients with Parkinson's disease (Bancher C, Lassmann H, Budka H, Jellinger K, Grundgke-Iqbal I, Iqbal K, Wiche G, Seitelberger F, Wisniewski H: J Neuropathol Exp Neurol 1:81, 1989), decorated in immunofluorescence microscopy Mallory bodies (MBs) present in livers of mice chronically treated with griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. In immunoblots it recognized very acidic MB components in a molecular weight range between 55 and 69.5 kilodaltons in addition to poorly soluble high molecular weight material. Moreover, an antibody to tau protein showed similar reactivities in immunofluorescence microscopy and immunoblotting experiments. Both antibodies also stained MBs in human liver with alcoholic hepatitis. These observations support and extend earlier findings which indicate that several intermediate filament-related cellular inclusion bodies, including MBs, share a variety of morphologic, structural and antigenic features. They also suggest the involvement of tau or tau-like proteins in MB formation.


Detection of interleukin-6 and alpha 2-macroglobulin immunoreactivity in cortex and hippocampus of Alzheimer's disease patients.

We examined brains from Alzheimer's disease (AD) patients by immunohistochemistry for the presence of protease inhibitors. Immunoreactivity for alpha 2-macroglobulin (alpha 2-M), the most potent of the known human protease inhibitors, was found in a subgroup of cortical and hippocampal AD senile plaques. In addition, large hippocampal neurons in AD brains displayed intracellular alpha 2-M immunoreactivity which was consistently stronger than in normal aged brains. In cultured human cells of neurogenic origin (SH-SY5Y neuroblastoma cells), alpha 2-M synthesis could be strongly induced by the inflammatory cytokine interleukin-6 (IL-6) indicating that human alpha 2-M behaves as an acute-phase protein in the nervous system. Therefore, we also examined AD brains for the presence of IL-6 and found strong immunostaining in and around a subgroup of senile plaques as well as around large cortical neurons. Only very few senile plaques also stained for C-reactive protein, an acute phase protein known to be inducible by IL-6. We propose that the presence of IL-6 and alpha 2-M immunoreactivity in AD brains is functionally linked and that a sequence of immunological events is part of the pathology of AD.


Prolonged monocyte accumulation in the lung during bleomycin-induced pulmonary fibrosis. A noninvasive assessment of monocyte kinetics by scintigraphy.

It has become more evident that monocytes, macrophages, and their products interact in a complex manner with various cell types in the lung, and may under the proper set of conditions contribute to the pathogenesis of pulmonary fibrosis. Current methods used to assess the lung content of mononuclear cells, which include tissue immunohistochemistry and bronchoalveolar lavage fluid analysis, sample the lung at one point in time and therefore provide only a "snapshot" of dynamic process. We utilized external imaging (scintigraphy) to provide a dynamic assessment of the trafficking patterns of radiolabeled monocytes in the lungs of rabbits in conjunction with lung tissue morphometry and bronchoalveolar lavage fluid analysis to determine the kinetics of neutrophil and monocyte accumulation in the alveolar walls and alveolar spaces of the lung during bleomycin-induced pulmonary fibrosis. We found that scintigraphy accurately reflected the accumulation of monocyte-associated radioactivity in the alveolar walls over time as well as the subsequent migration of these cells into alveolar spaces during the acute phase of bleomycin-induced lung injury (days 0 to 14) when compared with lung tissue morphometry. The scintigraphy, lavage, and morphometry data together showed that neutrophil influx into both of these lung compartments preceded that of monocytes by days, and that the influx of monocytes accounted for a major proportion of mononuclear cells found in the alveolar walls and alveolar spaces of the lung during this acute phase of inflammation. The increased numbers of neutrophils and mononuclear cells in alveolar spaces normalized by days 14 and 28 respectively, but in contrast to the normalization of neutrophil content in alveolar walls by day 10, increased numbers of mononuclear cells persisted in alveolar walls for up to 56 days, a time when there was a significant increase in the hydroxyproline content of these lungs. These data also show that the increased number of mononuclear cells present in the alveolar walls on days 28 and 56 was not due to a persistent influx of blood monocytes. These data suggest: (a) that differential pathways of efflux existed for alveolar wall versus alveolar space mononuclear cells, (b) that a delay in efflux from the alveolar walls occurred and/or that an increase in the local proliferation of mononuclear cells in this compartment may have been occurring during the later phases of bleomycin-induced lung injury, and (c) that this prolonged residence of mononuclear cells in the alveolar walls occurred concurrently with the development of pulmonary fibrosis.


Normal-hearing and hearing-impaired subjects' ability to just follow conversation in competing speech, reversed speech, and noise backgrounds.

The performance on a conversation-following task by 24 hearing-impaired persons was compared with that of 24 matched controls with normal hearing in the presence of three background noises: (a) speech-spectrum random noise, (b) a male voice, and (c) the male voice played in reverse. The subjects' task was to readjust the sound level of a female voice (signal), every time the signal voice was attenuated, to the subjective level at which it was just possible to understand what was being said. To assess the benefit of lipreading, half of the material was presented audiovisually and half auditorily only. It was predicted that background speech would have a greater masking effect than reversed speech, which would in turn have a lesser masking effect than random noise. It was predicted that hearing-impaired subjects would perform more poorly than the normal-hearing controls in a background of speech. The influence of lipreading was expected to be constant across groups and conditions. The results showed that the hearing-impaired subjects were equally affected by the three background noises and that normal-hearing persons were less affected by the background speech than by noise. The performance of the normal-hearing persons was superior to that of the hearing-impaired subjects. The prediction about lipreading was confirmed. The results were explained in terms of the reduced temporal resolution by the hearing-impaired subjects.


The role of dextran 40 and potassium in extended hypothermic lung preservation for transplantation.

We have previously demonstrated that a low-potassium dextran solution provides superior and more reliable preservation of lungs for 12 hours than that provided by the commonly used Euro-Collins solution. This study was designed to examine the individual contributions of dextran 40 and a low (extracellular) potassium concentration to lung preservation. In a randomized, blinded study using an in vivo canine single-lung transplant model, lungs preserved with low-potassium dextran solution (K+, 4 mmol/L; dextran 40, 20 gm/L) were compared to lungs preserved with low-potassium, no-dextran solution (K+, 4 mmol/L) and high-potassium dextran solution (K+, 123 mmol/L; dextran 40, 20 gm/L). The lungs were assessed immediately and 3 days after transplantation. The low-potassium dextran solution provided excellent immediate pulmonary function with little variability (arterial oxygen tension, 519 +/- 12 mm Hg, measured on the transplanted lung alone, inspired oxygen fraction = 1.0, n = 6). Removing the dextran 40 from the flush solution (low-potassium group) led to a significant deterioration in pulmonary function (arterial oxygen tension, 243 +/- 78 mm Hg, n = 6, p less than 0.01). The high-potassium dextran solution provided extremely poor preservation (arterial oxygen tension, 176 +/- 79 mm Hg; n = 6; p less than 0.01). Two animals in this group died within 6 hours of operation. Viability of the transplanted bronchus was significantly improved with the two solutions containing dextran 40. These results indicate that dextran 40 and low potassium concentration both contribute significantly to the uniformly excellent 12-hour lung preservation seen with the low-potassium dextran solution.


Improved lung preservation with dextran 40 is not mediated by a superoxide radical scavenging mechanism.

Improved lung preservation with a low-potassium dextran-containing solution has been previously demonstrated. In a subsequent study, it was shown that dextran 40 contributes significantly to this improved preservation. In the current in vitro study, human neutrophils suspended in lung preservation solutions (low potassium with dextran and low potassium without dextran) were stimulated to produce superoxide radicals. The presence of dextran in the solution did not significantly alter the amount of superoxide measured in the assay (low potassium with dextran, 4.149 +/- 0.144 nmol/10(6) cells/20 min; low potassium without dextran, 3.896 +/- 0.215; p greater than 0.2). This suggests that dextran 40 did not appreciably scavenge superoxide radicals, nor did it alter the production of superoxide radicals by stimulated neutrophils. Thus the significantly improved lung preservation seen with the use of dextran 40 is probably not mediated by a superoxide radical scavenging process.


Reoperation and mortality after surgical treatment of benign prostatic hypertrophy in a large prepaid medical care program.

The incidence of reoperation and mortality after prostatectomy was studied in 8,219 men who underwent surgical treatment for benign prostatic hypertrophy between 1976 and 1987 while they were members of the Kaiser Permanente Medical Care Program, Northern California Region. The vast majority (94.5%) received transurethral prostatectomy (TURP). The cumulative 8-year probability of a second prostatectomy was 7.6% after TURP and 2.1% after open prostatectomy. The risk of mortality associated with transurethral prostatectomy relative to open prostatectomy was 1.6 (95% confidence interval 1.2, 2.1) 8 years postsurgery. The increased risk of mortality associated with transurethral prostatectomy was most prominent during the first 5 years postsurgery (relative risk 1.8, 95% confidence interval 1.3, 2.5) and declined to 1.1 (95% confidence interval 0.8, 1.6) for deaths occurring after the first 5 years. The finding of an increased risk of mortality associated with transurethral prostatectomy is consistent with other studies and is unexplained.


The acute-phase response to turpentine-induced abscesses in malnourished rats at different environmental temperatures.

An assessment was made of the independent effect environmental temperature (13, 21, and 30 degrees C) and either protein deficiency or energy deficiency on the metabolic response of rats that had aseptic abscesses induced by subcutaneous injections of turpentine. Measurements of food intake, alpha 2-macroglobulin (alpha 2-M; a major acute-phase protein in the rat), albumin, and various circulating metabolites were made 48 hours after turpentine injection in animals acclimatized at 13, 21, and 30 degrees C and compared with pair-fed controls. Despite differences in basal circulating albumin concentrations between controls and protein deficient rats (P less than .001), turpentine produced a similar reduction in all groups of animals (approximately 10 g/L), independent of environmental temperature. The alpha 2-M response to turpentine was attenuated in all protein-deficient animals and also in the energy-restricted animals at 13 degrees C. The increase in circulating 3-hydroxybutyrate (BOH) and nonesterified fatty acid (NEFA) concentrations, which normally occur with reduced dietary intake, was reduced in the turpentine-injected animals to an extent that depended on prior dietary intake. It is concluded that the metabolic response, particularly the acute-phase protein response, to a standard form of "injury" is affected by protein deficiency and possibly by energy restriction under adverse environmental temperature.


Acetyl-coenzyme A carboxylase mRNA metabolism in the rat liver.

The acetyl-coenzyme A carboxylase (ACC) gene contains two promoters (PI and PII), both of which are active in the liver. Various physiological stimuli affect one, or both of the promoters of the ACC gene, and result in the generation of two classes of ACC mRNAs which differ in the composition of their 5' untranslated regions (5' UTR). We have analyzed the amounts of the two major mRNAs species that are generated from each of these promoters in order to examine the regulation of ACC gene activity in the liver under different physiological conditions. Our findings can be summarized as follows: (1) In liver from normal animals, fed a complete laboratory chow ad libitum, the level of class 2 ACC mRNA species generated by PII is very low. These mRNA species disappear on starvation. Refeeding starved animals with a fat-free diet stimulates both PI and PII with different time courses of induction: PII responds quickly and PII gene products accumulate to maximum levels within 18 hours, while the PI response, as measured by the accumulation of class 1 mRNAs, shows a lag period of 6 hours before reaching maximal levels at the end of a 24-hour refeeding period. The half-lives estimated from the induction kinetics were 4.4 hours for class 2 mRNAs and 11.8 hours for class 1 mRNAs. Reinstatement of starvation causes an almost instantaneous disappearance of class 1 mRNA species, as compared with class 2 mRNA species. This rapid decay of PI transcripts suggests that factors stabilizing this class of ACC mRNAs contribute to the steady-state levels reached after the dietary induction.(ABSTRACT TRUNCATED AT 250 WORDS)


Extreme heterogeneity of Ty1-copia group retrotransposons in plants.

We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants. All eight species studied contain reverse transcriptase fragments from Ty1-copia group retrotransposons. Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75%. Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila. The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants. Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species. We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.


Characterization of a UV-damage recognition factor in vitro that is associated with UV resistance in HeLa cells.

We have previously reported a cisplatin-selected HeLa cell line showing cross-resistance to ultraviolet (UV) radiation and overexpression of UV-damage recognition factors (Chao et al., Mol. Cell. Biol., 11, 2075-2080, 1991). Here, we further characterize a UV-damage recognition factor in vitro using a gel mobility shift assay. The results indicate that the damage-recognition factor is (i) localized mostly in the nucleus, (ii) protease-sensitive, (iii) RNA-independent, (iv) active in a wide range of ionic strengths (50-400 mM NaCl), (v) with a high affinity for UV-damaged DNA (50-fold molar excess competitor causes 50% recognition loss), and (vi) resistant to agents and that modify protein conformation (urea and NP-40), but slightly sensitive to CaCl2. The significance of the identified UV-damage recognition factor in the sensitivity or resistance of cells to UV is also discussed.


Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection.

We have compared cytoplasmic- and nuclear-delivered, glass needle-mediated microinjection protocols for their ability to support both transient and stable phenotypic expression of reporter gene constructs in non-immortalized human skin fibroblasts cultures. Microinjection of form I (covalently closed circular, supercoiled) plasmid pMC38 DNA into the nucleus of human cells resulted in high levels of transiently expressed p110gag-myc oncoprotein as detected by immunofluorescence microscopy. Likewise, the nuclear delivery of a plasmid construct bearing the entire simian virus 40 genome induced the formation of morphologically transformed foci in approximately 6% of the recipient cell population. In contrast, the introduction of plasmid DNA by the cytoplasmic route proved virtually incapable of supporting either transient gene expression or morphological transformation. In situ autoradiography of cells injected with 3H-labelled plasmid DNA revealed that whereas the material delivered directly into the nucleus was retained by this subcellular compartment for prolonged times (greater than or equal to 48 h), the radiolabelled DNA molecules introduced via the cytoplasmic route did not reach the nucleus and appeared to be substantially degraded within 8 h following injection. These results indicate unequivocally that nuclear injection is the route of choice when monitoring foreign gene expression in human cells.


Overproduction of the RecD polypeptide sensitizes Escherichia coli cells to gamma-radiation.

We investigated DNA metabolism in Escherichia coli cells carrying the multicopy recD+ plasmid (pKI13). In the presence of pKI13, the cellular level of the recD gene product (RecD polypeptide) is amplified at least 60-fold. Overproduction of the RecD polypeptide has no effect on UV repair and conjugational recombination. In contrast, high cellular levels of this polypeptide sensitize wild-type cells to gamma-radiation; also, they increase the rate of radiation-induced DNA degradation. A possible mechanism for the enhancement of gamma-ray-induced killing by large amounts of the RecD polypeptide is discussed.


The persistence of sister-chromatid exchange frequencies in men occupationally exposed to vinyl chloride monomer.

The persistence of sister-chromatid exchange frequencies in a population occupationally exposed to the well known chemical mutagen vinyl chloride monomer was studied. It was shown that increased values of sister-chromatid exchange frequencies were still present in the lymphocytes of workers who had not been exposed for 8-120 days and retired persons for 5-10 years after exposure. The possible ability of vinyl chloride monomer alkylating metabolites to cause long-lasting damage of the DNA molecule is discussed.


No increase in chromosome aberrations in lymphocytes from workers exposed to nitrogen fertilisers.

The putative genetic risk of people occupationally exposed to nitrogen fertilisers was studied using the structural chromosome aberration assay in peripheral blood lymphocytes. The exposed group included 23 subjects working at complex and mixed fertiliser plants. The percent of aberrant cells (Ab.C %) and break to cell ratio (B/C) were 0.95% and 0.01 respectively. The matched control group (20 subjects) was found to have 0.80% Ab.C and a B/C ratio of 0.0085. The results show a lack of detectable genetic damage in exposed people using this cytogenetic approach.


SOS system induction in Escherichia coli cells with distinct levels of ribonucleotide reductase activity.

The UV-mediated induction of recA and sfiA genes in Escherichia coli cells with distinct levels of dATP has been studied. Low levels of dATP were obtained by using either a temperature-sensitive ribonucleotide (RDP) reductase-deficient (nrdA) mutant or a wild-type strain treated with hydroxyurea. High pools of dATP were achieved by using a plasmid overproducing RDP reductase. The results obtained show that expression of the recA and sfiA genes was inhibited neither in the UV-irradiated nrdA mutant at 42 degrees C nor in the wild-type strain in the presence of hydroxyurea. Likewise, the increase of the dATP pool did not enhance recA and sfiA gene expression after UV irradiation. All these data suggest that the basal level of dATP is not a limiting factor in the process of induction of the SOS system in Escherichia coli.


Mutagenicity of pyrrolizidine alkaloids in the Salmonella typhimurium/mammalian microsome system.

The mutagenicity of a series of pyrrolizidine alkaloids, and of extracts from several Italian Senecio species containing pyrrolizidine alkaloids, including S. inaequidens, S. fuchsii and S. cacaliaster, were tested using the Salmonella typhimurium/mammalian microsome system. Retrorsine, senecivernine, seneciphylline and the Senecio extracts showed a weakly mutagenic activity.


The protective role of polyphenols in cytotoxicity of hydrogen peroxide.

A variety of synthetic and natural polyphenols protect mammalian cells from hydrogen peroxide (H2O2). Cytotoxicity of H2O2 on Chinese hamster V79 cells was assessed with a colony formation assay, and several polyphenols prevented the decrease in the number of colonies caused by H2O2. A study of the structure-activity relationship revealed that affinity of the polyphenols for the cell membrane and the presence of an ortho-dihydroxy moiety in their structure proved essential to this protection.


Frameshift mutagenesis in bacteriophage T7.

We have undertaken an initial characterization of frameshift mutagenesis in bacteriophage T7 and have identified a subset of very low reversion frameshift mutations in the T7 ligase gene (gene 1.3). We used this information to construct bacteriophage T7 strains that contain one extra or one less base pair in gene 1.3 such that a frameshift event restores the reading frame of that gene. These events can be quantified and the frameshift mutation isolated within a localized region of the ligase gene. We have also identified a portion of the T7 ligase protein that will accept tracts of nonsense amino acids yet still give a ligase positive phenotype. This allows flexibility in the design of the target DNA sequence with which to study frameshift mutagenesis. These assays for frameshift mutagenesis performed in E. coli cells infected with the appropriate T7 strain, were used to measure the frequency of both plus and minus frameshifts in vivo.


The effect of lead on Allium cepa L.

The effect of lead on Allium cepa L. at concentrations of 0.1, 1.0, 10, 50, 100 and 200 ppm were studied. Analysis focused on root growth, frequency of mitosis in a meristematic zone, and chromosomal aberrations. It was observed that lead reduces root growth and the frequency of mitotic cells in meristematic zones, and increases the frequency of aberrant cells. The intensity of the effects is a function of lead concentration.