Interferon-gamma induces phosphorylation of multiple small-molecular-weight proteins in U937 cells. This study examines the hypothesis that interferon-gamma (IFN-gamma) induces protein phosphorylation as part of the signal transduction pathway used to activate U937 cells. U937 cells labeled with 32Pi were treated with IFN-gamma, proteins were separated by two-dimensional polyacrylamide gel electrophoresis, and the pattern of protein phosphorylation was determined by autoradiography and computer-assisted two-dimensional densitometry. IFN-gamma (100 U/ml) induced phosphorylation of multiple proteins between 15 and 60 min, and the proteins were all dephosphorylated by 120 min. The pattern of proteins phosphorylated in the presence of ionomycin or PMA differed from that of IFN-gamma. Inhibition of protein kinase C activity by 1-(5-isoquinolinesulfonyl)2-methyl piperazine (H-7), inhibition of calcium-calmodulin-dependent protein kinase by N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7), and inhibition of calcium redistribution by 8-(diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) did not inhibit the majority of IFN-gamma-induced protein phosphorylation. These data indicate that IFN-gamma induces protein phosphorylation in U937 cells by activation of a kinase different from, or in addition to, protein kinase C or calcium-calmodulin-dependent kinase.


Interferon induction by viruses. XXI. Vesicular stomatitis virus: interferon inducibility as a phylogenetic marker. Forty-five vesiculovirus isolates were systematically compared for their capacity to induce interferon (IFN) in chick embryo cells under conditions such that the maximum (quantum) yield of IFN per cell and the titer of IFN-inducing particles (IFP) could validly be determined. Twelve isolates of the New Jersey (NJ) serotype of vesicular stomatitis virus (VSV) were good inducers, yielding amounts of IFN that ranged in a continuum from 300 to more than 8,000 units per 10(7) cells. These must reflect genetic differences between the closely related viruses. These differences were not reflected in the nucleotide sequence of the viral 3' leader RNA, for analysis of eight of the NJ isolates showed no correlation with the IFN yields. As found in previous smaller surveys, 28 out of 32 VSV isolates of the Indiana (IN) serotype produced little or no IFN, or even suppressed its induction. However, four exceptional IN strains were isolated during 1984 and 1985 from cattle within a relatively circumscribed geographical area in Costa Rica and Panama; all belonged to Indiana virus, type 1, subtype IV, in the proposed G-protein gene evolutionary tree. This is the first example of an IFN-inducing phenotype serving as a phylogenetic marker.


Anti-HIV-1 activity of recombinant and hybrid species of interferon-alpha. To identify candidate interferons (IFNs) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection and to investigate sequence-function relationships, the antiviral activities of nine species of recombinant IFN-alpha [IFN-alpha A, IFN-alpha B, IFN-alpha C, IFN-alpha D, IFN-alpha J, [Ser116]IFN-alpha J1, IFN-alpha K, IFN-alpha J/C(Fnu4HI), and IFN-alpha A/D(BglII)] were evaluated against HIV-1. MT-2 cells were exposed to various concentrations of each IFN and were then infected with HIV. Protective effect was determined by cell viability using a tetrazolium dye assay. Activity against vesicular stomatitis virus (VSV) was assessed on MDBK and WISH cells. The 50% inhibitory concentration against HIV was 37 +/- 14 pg/ml for IFN-alpha A, and ranged from 15 +/- 3 pg/ml for IFN-alpha J/C(Fnu4HI) to > 90,000 pg/ml for IFN-alpha D. In general, relative activity against HIV was similar to relative activity against VSV on WISH cells. IFN-alpha D was notable for its decreased activity on human cells. The observations suggest that it may be possible to produce IFNs-alpha with more favorable therapeutic indices than currently available IFNs. Furthermore, the anti-HIV activity of IFNs-alpha is not determined solely by their linear amino acid sequence.


[Serotypes and electropherotypes of group A human rotaviruses in patients with acute gastroenteritis in Saitama area, 1988-1991]. Serotypes and RNA electropherotypes of group A human rotaviruses were identified in stool samples from patients with acute gastroenteritis in Kasukabe Kosei Hospital, Saitama, Japan, during three rotavirus seasons from 1988 to 1991. Of the 665 stool samples from patients with acute gastroenteritis, 169 (25.4%) stool samples were positive for group A human rotaviruses. Of these 169 samples, 98 (58.0%) were serotype 1, 7 (4.1%) serotype 2, 14 (8.3%) serotype 3, 3 (1.8%) serotype 4 and 47 (27.8%) were untypable. Serotype 1 was predominant over all three rotavirus seasons. Serotype 2 was most prevalent in the second rotavirus season, serotype 3 in the third season. Serotype 4 was detected only the second rotavirus season. The ratio of subtype I to subtype II was 4.5:95.5. Among the 162 strains examined, 139 specimens were available to be electropherotyped. Fifteen different RNA electropherotypes were detected. Three to four different electropherotypes were more prevalent in each rotavirus season and the detection rate in each year was different within the same electropherotypes. The rotavirus strains of the same electropherotypes were observed with high prevalence for every rotavirus seasons. The same electropherotypes was found in human rotavirus strains of different serotypes that were appeared at different epidemic seasons.


[Immunization of healthy children with measles-mumps-rubella trivalent vaccine simultaneously given with varicella vaccine]. Trivalent vaccine, containing measles TD97, rubella TCRB-19 and mumps NK-M46 strains (MMR vaccine) was administered to a total of 116 healthy children of which 50 subjects were simultaneously injected with varicella vaccine in the opposite arm. The seroconversion rates for measles, mumps, rubella, and varicella in those who received both MMR and varicella vaccines (MMR + V group) were 100% (44/44), 91% (39/43), 100% (46/46) and 95% (39/41), respectively. And these rates were comparable to those in subjects receiving only MMR vaccine, namely 100% (64/64) for measles, 95% (57/62) for mumps, and 97% (58/60) for rubella. Fifty-eight children receiving MMR vaccine were seronegative to measles, mumps and rubella before vaccination, and 51 (88%) of them were found to be seropositive against all three viruses at 6 to 8 weeks after vaccination. Among the children injected with MMR and varicella vaccines, 36 subjects had no pre-antibodies to measles, mumps, rubella and varicella. Seroconversion in post-serum to all four viruses were found in 31 cases (86%). Clinical reactions observed in some vaccines were mild fever (17%) and exanthem (5%). There were no complications of lymphadenopathy, swelling in parotid regions, or meningitis. Our results indicate that simultaneous administration of MMR vaccine and varicella vaccine is a safe and effective method for immunizing children against these four infectious diseases.


[Family-acquired hepatitis A--prevalence of hepatitis A among the family in Aichi Prefecture, 1990]. We studied the transmission of hepatitis A virus (HAV) in 45 families, which members were diagnosed as hepatitis A in 8 hospitals in 1990. Feces and sera from 50 patients and their 126 family members were tested for HAV-specific antigen and IgM antibody by ELISA or polymerase chain reaction (PCR) technology. From the interval of the onset of hepatitis or detection of HAV antigen in feces, HAV transmission was recognized in 11 (24.4%) of 45 families. The transmission was found to be concerned with contacts of the children and that from children to parents was found in 4 families and the reverse in 2. HAV antigen was detected from feces of 4 family members before onset of icterus by ELISA and furthermore, 3 by PCR. It was indicated that these methods would be used to prevent the transmission in a family, day-care centers, or institutions for the mentally retarded.


[Three cases of lower respiratory tract infection worsened after rhinovirus infection]. We experienced three cases of lower respiratory tract infections worsened after Rhinovirus infection. Case 1: A 42-year-old male with diffuse panbronchiolitis was admitted to our hospital with the complaint of dyspnea on November 21, 1988. Rhinovirus was isolated from nasal washing and P. aeruginosa was cultured from transtracheal aspiration (TTA). Case 2: A 67-year-old male, whose underlying disease was pulmonary asbestosis, was admitted to our hospital complaining of pyrexia on June 12, 1990. Rhinovirus was isolated from TTA and H. influenzae and others were cultured from TTA. Case 3: A 64-year-old male with pulmonary emphysema was admitted to our hospital with a complaint of dyspnea on August 11, 1989. On December 17, 1989 the patient developed rhinorrhea and complained of purulent sputum, pyrexia and dyspnea after five days. Rhinovirus was isolated from nasal washing and TTA and S. nonhaemolyticus and others were cultured from TTA. As indicated in this report, it is interesting to study the relationship between viral infection of the upper respiratory tract and bacterial infection of the lower respiratory tract.


[Outbreaks of acute gastroenteritis caused by small round structured viruses in Tokyo]. Of 34 non-bacterial gastroenteritis outbreaks which occurred at day-care centers, kindergartens, elementary and secondary schools in Tokyo during the period from February 1985 to June 1991, 28 outbreaks from which small round structured viruses (SRSV) were detected in the patients' stool specimens by electron microscopy were subjected to an epidemiological investigation. The outbreaks tended to occur frequently in the cold season; twenty-two (79%) of these outbreaks from November through April. Though detailed epidemiological informations was not obtained from all outbreaks, the common source of infection were presumed to be present in many of the outbreaks, judged from the incidence as to time course of patients. Food doubted to be incriminated as transmission vehicles in these outbreaks was served at schools, kindergartens, and lodgings. In some outbreaks, SRSV was detected from stool specimens of food handlers, or they were seroconverted to SRSV, suggesting that food was incriminated as a transmission vehicle. The symptoms of patients differ slightly from age to age: in the age range of 0 to 6 years, vomiting 90%, fever 41% and diarrhea 32%; in the 6 to 12 year-olds, nausea 61%, vomiting 48%, abdominal pain 65%, diarrhea 20% and fever 29%; and in the 12 to 15 year-olds, nausea 69%, vomiting 42%, abdominal pain 60%, diarrhea 30% and fever 34%. The lower the age of patient vomiting was more frequently observed. In these lower age groups, the frequency of nausea and vomiting tended to exceed that of diarrhea.


[Evaluation of micro particle enzyme immunoassay technique (MEIA)-IMx for the detection of antibody to hepatitis A virus]. A new micro particle enzyme immunoassay technique (MEIA, IMx HAVAB, Abbott) has been recently introduced for the detection of antibody to hepatitis A virus (anti-HA). To evaluate the feasibility of the IMx HAVAB, we carried out comparison tests between MEIA, RIA and EIA. Furthermore, we investigated the prevalence of anti-HA in Fukuoka City and Yonaguni Island, Okinawa, Japan using this method. Results obtained were as follows: In the test of 514 sera, 254 (49.0%) were positive by the three methods, and the remaining 260 (50.6%) were negative by the three methods. Examination of diluted sera using IMx revealed that IMx can be used as efficiently as RIA and EIA. Quantitative linear correlations were found between IMx and RIA (r = 0.973), and IMx and EIA (r = 0.969). Anti-HA was assayed within 45 minutes by IMx, and the procedure was simple because of the auto analyser used in this method. On Yonaguni Island, a significant decrease in the overall prevalence of anti-HA from 75.2% in 1980 to 61.1%, in 1990 (p less than 0.05) was found, the prevalence of anti-HA on Yonaguni Island in 1990 was significantly higher than in Fukuoka City (p less than 0.001). These results indicate that the sensitivity of IMx is equivalent to those of RIA and EIA, that it is easier to use than either RIA or EIA and that hepatitis A virus infection in Okinawa, has significantly decreased during the past 10 years, but is still significantly more frequent than in Fukuoka City.


[Effect of azithromycin on human serum sensitivity of Pseudomonas aeruginosa]. We examined the effect of azithromycin (AZM), a 15-membered azalide newly synthesized from erythromycin (EM), on serum sensitivity of 6 strains of Pseudomonas aeruginosa. Incubation for 48 h on agar with EM 12 micrograms/ml or AZM 1.6 micrograms/ml induced increased serum sensitivity in 2 of 6 strains (S-6, PA-103), but there were no changes in any strains with josamycin (JM) 12 micrograms/ml. Although EM 12 micrograms/ml induced increased serum sensitivity of S-6 after more than 36 h incubation, AZM 1.6 micrograms/ml induced increased serum sensitivity of this strain at 12 h incubation. AZM 0.8 microgram/ml (1/62.5 MIC) showed more potent activity to enhance serum sensitivity of S-6 than that of EM 12 micrograms/ml (1/8 MIC) after 48 h incubation. P. aeruginosa S-6 incubated with EM 12 micrograms/ml or AZM 1.6 micrograms/ml for 48 h was less hydrophobic than that of control bacteria, but there was little change in the hydrophobicity of the strain incubated with JM 12 micrograms/ml. These results show that AZM has more potent activity to enhance serum sensitivity of P. aeruginosa than that of EM. Since decrease of cell surface hydrophobicity of P. aeruginosa S-6 was correlated with increased serum sensitivity, EM and AZM may induce enhanced serum sensitivity by changing cell surface structure of P. aeruginosa.


[Clinical study of bioabsorbable PGA sheets for suture reinforcement and use as artificial pleura]. Bioabsorbable PGA non-woven fabric sheets were used to treat 103 patients who underwent pulmonary surgery in three hospitals, and their handleability, applicability, drainage time after surgery and subsequent side effects were studied. For suture reinforcement, these sheets showed satisfactory handleability, applicability and effectiveness for hemostasis and prevention of air leakage at the suture sites. Since this material has good compatibility with fibrin glue, use of these two materials in combination reduced both the operation time and postoperative drainage period. For small fistula and pleural defects, attachment of the sheets with fibrin glue to create an artificial pleura was sufficient for prevention air leakage without suturing. No side effects or complications were observed, and the postoperative courses of all 103 patients were uneventful. These findings suggest that PGA sheets are acceptable for suture reinforcement in the pulmonary surgery, and that when used with fibrin glue, can simplify surgery for emphysematous lung disease and shorten the period of postoperative air leakage.


[Phrenic nerve palsy after open heart surgery--problems in post-operative respiratory management]. We clarified the problems in respiratory management in patients with phrenic nerve palsy (PNP) after open heart surgery. From December 1988 to March 1991, 248 adult patients underwent open heart surgery with topical myocardial cooling. In these patients PNP was diagnosed in 17 patients (6.9%). Age of these patients at operation ranged from 25 to 76 years with a mean age of 57 years. 9 patients were valvular heart disease (7 were reoperation), 7 were coronary artery disease, and one patient was aortic aneurysm. PNP was diagnosed by chest roentgenogram or percutaneous phrenic nerve stimulation test (PNST). Frequency of long-term intubation (> 14 days) was higher in complete PNP patients than incomplete PNP patients. 2 patients, who were observed effort dyspnea at the time of respiratory weaning, were extubated followed by respiratory and circulatory deterioration, and were reintubated. PNP were confirmed after reintubation by PNST. These patients died of pneumonia. Another 2 patients were observed effort dyspnea during respiratory weaning, and were doubted of PNP. PNP were confirmed by PNST before extubation. After hemodynamic stabilization and respiratory physiotherapy, these patients were extubated without any trouble. One patient, who required repeated intubation, was diagnosed PNP by PNST. This patient was extubated after confirmation of improvement of PNP by follow-up PNST. The incidence of PNP was higher in patients with previous open heart surgery than without previous operation. We supposed that the dissection around the heart might be one of major cause of postoperative PNP. So, in recent 4 cases of mitral reoperation, we attempted to reach mitral valve through atrial septum with minimum dissection.(ABSTRACT TRUNCATED AT 250 WORDS)


[Malignant fibrous histiocytoma of the mediastinum associated with bilateral adrenal gland metastases--a case report]. A 55-year-old female underwent complete resection of left posterior mediastinal tumor and both adrenal gland tumors. Histological examination revealed malignant fibrous histiocytoma (MFH) showing a storiform pattern. Her postoperative course was uneventful and well one year after the operation. We concluded the tumor arose from the mediastinum and metastasized to the bilateral adrenal glands. No other case was reported that primary MFH of the mediastinum with distant metastases was completely resected including metastatic lesions.


[Malignant fibrous histiocytoma of the heart]. A 59-year-old woman was referred to our hospital by a local cardiologist, with unknown cause of congestive heart failure and respiratory failure. No significant heart murmur was heard and a cardioechogram from the anterior chest was insufficient for diagnosis. On the night of admission, cardio-respiratory shock was found, and Swan-Gantz catheter data showed severe pulmonary hypertension. Transesophageal echo-cardiography (TEE) revealed a left atrial tumor, with wide neck anchored onto the posterior wall of the left atrium, and which was floating into the left ventricle during the diastolic phase. An emergency operation was performed and the tumor, (80 by 50 by 48 mm) was resected. The patient recovered well in the postoperation period. However the residual tumor continued to grow gradually in the pericardial cavity and the patient died 3 months after the operation. Primary MFH of the heart is rare and 29 cases have been reported. The prognosis of MFH is poor despite surgery. Whenever a left atrial tumor with a wide neck is attached to the posterior wall, it must be considered to be possibly malignant and surgical resection is recommended.


Neuroendocrine-immune interactions associated with loss and restoration of immune system function in spinal cord injury and stroke patients. Both natural and adaptive immune responses were shown to be strikingly decreased in initial blood samples from 34 spinal cord injury and stroke patients. NK-cell function decreased to 24.8% (mean) 2 weeks after spinal cord injury in previously healthy young adults whose control group revealed a mean NK-cell function of 48.7%. This was accompanied at 2 weeks by increased plasma ACTH (mean of 17.0 pg/ml from 17 patients compared to a mean of 11.2 pg/ml from 12 controls) and urine free cortisol levels (mean of 152.1 micrograms/24 h from 9 patients compared to 53.6 micrograms/24 h from 15 controls). T-cell function and/or activation decreased to below normal values within 3 months after injury as revealed by lymphocyte transformation that was 32.8% of normal at 3 months. T-cell activation diminished as shown by a mean IL-2 receptor level of 179.3 units/ml in patients compared to 328.2 units/ml in controls. Serial monitoring of NK- and T-cell function revealed that specific physical rehabilitation therapy over a period of 6 months after injury restored NK- and T-cell function to near normal levels in most patients. This improvement was accompanied by a parallel rise in the patient's functional independence measurement scores. Results suggest critical neuroendocrine-immune system interactions in the restoration of immune function. Cortisol levels reverted to normal after 6 months of rehabilitation. Limited data suggest that natural immune system depression, NK-cell function, persists in spinal cord injury patients not receiving rehabilitation therapy (mean NK-cell lysis of 10.3%; p < 0.01).


Tumor necrosis factor receptors--structure and function. Tumor necrosis factors (TNFs) have been a focus of research for well over a decade now. The identification and recent molecular cloning of two different types of cell-surface TNF receptors will shed further light on the mode of action of these pleiotropic cytokines. In the present article, we summarize the data on the biochemistry and structure of the receptors and focus on the molecular cloning of the respective cDNAs. The nucleotide sequences of the receptor genes revealed that both TNF receptors belong to the still growing nerve growth factor receptor gene family. The function and origin of TNF inhibitory proteins as well as receptor-mediated signal transduction are discussed.


[The positive rate of hepatitis C virus antibody detected by the second generation method in pregnant women and influence of pregnancy and delivery on HCV infection]. The C100-3 antibodies to HCV can be examined with a Chiron kit but only the antibody to non-structure-4 can be. The antibodies in sera of pregnant women were examined by a passive hemagglutination method (PHA), which is one of the second generation kits used in detecting antibodies to the hepatitis C virus. We studied the percentage of pregnant women in our hospital with antibodies to HCV, and tried to compare the C100-3 antibodies with PHA results in pregnant women. The results are as follows. 1) In the sera of 235 pregnant women both the PHA antibody and the C100-3 antibody were examined. Two women (0.85%) had the C100-3 antibody and 4 (1.7%) had the PHA antibody. 2) Two women positive for both PHA and C100-3 antibodies were positive for PCR during pregnancy. But 2 women positive for PHA only were negative for PCR during pregnancy and positive after delivery. So 4 women positive for PHA had HCV RNA. 3) Next, the sera of 1,198 pregnant women were examined by the PHA method, and nine women were positive. Only four of the 9 were positive for C100-3. 4) It is speculated that the positive rate for anti-HCV antibodies in the sera of pregnant women in Nagoya city is 0.75%. 5) There were 3 cases that were negative for PCR during pregnancy and positive after delivery. So it is speculated that HCV can be activated after delivery.


Changing trends in treatment of substance abuse: a look at some of the factors involved in third-party providers and treatment planning. In order to better focus our care in providing patients with treatment that matched their needs, an inpatient study was done on patient discharges for 1990 involving 267 patients. Our study points out the influence of the changing drug scene in this country. In approximately 40% of the patients admitted, the drug of choice is other than alcohol. The importance of family and employer involvement in effective treatment stands out. Fewer patients are being admitted but those admitted are sicker and require more treatment. Chemical dependency programs in Louisiana and the United States are experiencing significant declines in the utilization of their service and many are closing. The patient census has been impaired primarily by the expense of or lack of insurance coverage by third-party payors. For patients with insurance coverage the criteria for inpatient treatment by third-party reviewers are so stringent that staff assessment recommendations are frequently over-ruled. Substance abuse is a chronic relapsing illness, yet many insurance companies limit coverage both in time and money. In no other field of medicine do patients leave treatment prematurely as they do in substance abuse.


Different guanosine triphosphate-binding proteins couple vasopressin receptor to phospholipase C and phospholipase A2 in glomerular mesangial cells. To evaluate the identity of the guanosine triphosphate--binding proteins coupling arginine vasopressin receptor occupancy with activation of phospholipase C, leading to Ca2+ mobilization, and activation of phospholipase A2, leading to arachidonate release and prostanoid formation, we used intact cells, saponin-permeabilized cells, and membranes of the rat mesangial cell. Arginine vasopressin 10(-7) mol/L produced a dose-dependent increase in cytosolic Ca2+ to maximal levels of 500 nmol/L with peak responses occurring within 10 seconds of addition of arginine vasopressin to cells in suspension. Arginine vasopressin 10(-7) mol/L elicited a maximal response. These increases were associated temporarily with a fourfold increase in tritiated D-myo-inositol 1,4,5-trisphosphate formation in prelabeled cells. Pertussis toxin (200 ng/ml) did not inhibit the Ca2+ increase nor did it inhibit the increase in tritiated D-myo-inositol 1,4,5-trisphosphate formation, suggesting a pertussis toxin--insensitive signaling pathway for phospholipase C hydrolysis in response to vasopressin. Membranes prepared from mesangial cells increased D-myo-inositol 1,4,5-trisphosphate formation in vitro in response to arginine vasopressin and guanosine-5'-0(3- thiotrisphosphate), and this stimulation was inhibited by guanosine-5'-0(2-thiodiphosphate), confirming the involvement of a guanosine triphosphate--binding protein. In contrast arginine vasopressin stimulated arachidonate release from intact mesangial cells, and this effect was blocked by pretreating cells with pertussis toxin. To demonstrate that this was through a pertussis toxin--sensitive guanosine triphosphate--binding protein, we permeabilized cells with saponin and determined that arginine vasopressin and guanosine-5'-0(3-thiotriphosphate) stimulated the release of arachidonic acid and the stimulation of guanosine-5'-0(3-thiotriphosphate) was inhibited by guanosine-5'-0(2-thiodiphosphate). Finally, pertussis toxin was able to stimulate adenosine diphosphate ribosylation in vivo of a substrate protein in mesangial cell membranes of 41 kd, and this ribosylation was inhibited by pretreating cells with pertussis toxin. These data suggest that the release of arachidonic acid by vasopressin in glomerular mesangial cells is linked to a pertussis toxin--sensitive guanosine triphosphate--binding protein and that this activation of phospholipase C in vasopressin is linked to a pertussis toxin--insensitive guanosine triphosphate--binding protein.


Lipoxin-induced migration of fish leukocytes. The migration-inducing abilities of both synthetic lipoxin A4 (LXA4) and leukotriene B4 (LTB4) for rainbow trout neutrophils were examined with an in vitro assay. LXA4 caused enhanced migration of these cells with a three- to fourfold greater response than that observed toward LTB4 at most concentrations tested. Checkerboard assays showed that synthetic LXA4 was chemokinetic for trout leukocytes, while LTB4 was chemotactic. The chemokinetic ability of LX synthesized by rainbow trout macrophages maintained in short-term culture was also determined. These cells produced both 4-and 5-series LX, namely LXA4, LXA5, 11-trans-LXA4, 11-trans-LXA5, 7-cis-11-trans-LXA4 and 6(S)-LXA4. Although greater migration was found to LXA4 than to most of the other 4-series positional isomers, at least at some concentrations, this did not exhibit the degree of stereospecificity previously reported in some studies for mammalian leukocytes. Little difference was found between the chemokinetic responses of rainbow trout leukocytes to 4- and 5-series LX. These findings suggest that LXs are important migration-inducing molecules in trout and have a relatively long evolutionary history.


Prostaglandin E2 receptor activity and susceptibility to natural killer cells. We have described a high-affinity receptor for prostaglandin E2 (PGE2) present on metastatic murine mammary tumor cells. Pharmacologic antagonism of this receptor increases metastatic potential. In the present study, we have asked whether the binding activity of PGE on tumor target cells plays a role in natural killer (NK)-target cell interactions. We have used three unrelated PGE-receptor antagonists, SC19220, LEO101, and AH6809, to show inhibition of [3H]PGE2 binding to YAC-1 cells and inhibition of PGE2-mediated elevation of intracellular cyclic AMP (cAMP). Addition of any of these three receptor antagonists to standard 4-h 51Cr-release assays inhibits YAC-1 lysis by NK-enriched populations from murine spleen. This is the first report that antagonism of PGE binding affects NK activity. Our studies demonstrate that these effects are mediated through inhibition of target-effector cell conjugate formation. Studies in which effector and target cells were pretreated separately indicate that the PGE-mediated effects are expressed at the target cell level.


Interferon-alpha-dependent and -independent participation of accessory cells in natural killer cell-mediated lysis of HSV-1-infected fibroblasts. Human natural killer (NK) cells require an HLA-DR+ accessory cell (AC) population to lyse herpes simplex virus-type 1 (HSV-1)-infected fibroblasts (HSV-Fs) but not K562 target cells. It has been postulated that ACs may function by producing interferon-alpha (IFN-alpha), which stimulates NK cells. Using a sequential enrichment protocol, ACs were found to coenrich with the interferon-producing cells (IPCs). Treatment of the ACs with a protein synthesis inhibitor, emetine, inhibited both their IFN production and AC function, results that support a central role for IFN in AC activity. In contrast, when the arginine analogue canavanine was added to NK assays, no IFN-alpha was produced and NK(HSV-Fs) activity was only partially inhibited. Consistent with IFN-independent AC function, treatment with either polyclonal sheep or bovine anti-IFN-alpha neutralized all the IFN-alpha produced during the NK assays but caused either no or partial reduction of NK(HSV-Fs) activity, respectively. However, when limiting numbers of ACs were used, the bovine antiserum neutralized both IFN-alpha and NK(HSV-Fs) activity. We further found that HLA-DR+ cells are required for cell clustering, suggesting a role for cell contact. Finally, fixation of activated ACs prevented the accessory function. Together, these results demonstrate that ACs can provide help to NK cells in both IFN-alpha-dependent and -independent manners.


Mepacrine inhibits fMLP-induced activation of human neutrophil granulocytes, leukotriene B4 formation, and fMLP binding. Pulmonary sequestration and activation of polymorphonuclear leukocytes (PMNLs) are characteristic of many forms of acute lung injury. The present experiments were designed to study the effects of mepacrine on human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (fMLP). Mepacrine inhibited fMLP-induced superoxide production and degranulation in a dose-dependent manner with Kd values of 2.3 +/- 0.5 x 10(-7) M and 5.7 +/- 1.3 x 10(-6) M, respectively. Stimulation of PMNLs by 10(-6) M fMLP provoked the formation of barely detectable amounts of leukotriene B4 (LTB4) (< 5 pg/10(7) cells). Pretreatment of the cells with cytochalasin B augmented generation of LTB4 in response to fMLP (339 +/- 79 pg/10(7) cells). LTB4 formation was also inhibited by mepacrine (50% inhibitory concentration 1.0 +/- 0.5 x 10(-6) M). Furthermore, mepacrine inhibited the specific binding of [3H]fMLP to neutrophils with a Ki value of 1.4 +/- 0.4 x 10(-5) M. Mepacrine decreased the receptor binding affinity without altering the number of receptors. These findings demonstrate that the inhibitory effect of mepacrine is response dependent and suggest that this action of mepacrine could, in part, be attributed to a decrease in fMLP receptor affinity.


Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism. In primary cultures of rat hepatocytes, prostaglandin E2 and prostaglandin D2 (PGE2 and PGD2) inhibited the secretion of very low density lipoprotein (VLDL)-associated apoB, triacylglycerol, and cholesterol. These effects were concentration-dependent and remained apparent for at least 3 days of culture without an effect on the apoB/triacylglycerol ratio of the secreted VLDL. Prostaglandins had no effect on the overall synthesis of triacylglycerol but triacylglycerol accumulated within the cells, without intracellular accumulation of apoB. PGE2, when added to the medium together with glucagon, increased the inhibition of VLDL secretion, compared to that observed with glucagon alone. However, PGE2 did not increase the stimulatory effect of glucagon on ketogenesis. Unlike glucagon, the prostaglandins did not inhibit fatty acid synthesis nor did they stimulate ketogenesis or production of cAMP. Thus, of all the parameters of hepatic lipid metabolism studied, PGE2 and PGD2 selectively affected VLDL. Selective inhibition of VLDL secretion was also observed with the calcium antagonist verapamil. The divalent cation ionophore A23187 also inhibited VLDL release but, in contrast, also inhibited fatty acid and cholesterol synthesis. The results suggest that VLDL secretion is modulated at some optimal cell calcium concentration that may be mediated selectively by agents such as prostaglandins.


Accumulation and mobilization of triglycerides and cholesteryl esters in Leydig tumor cells. Incubating MA-10 Leydig tumor cells with sodium oleate led to the accumulation of triglyceride within the cells. Triglycerides were deposited in a time- and dose-dependent fashion. Cellular triglyceride promoted storage of cholesteryl ester. As much cholesteryl ester was stored in oleate-treated cells as in cells treated with saturating concentrations of low density lipoprotein. Addition of both oleate and low density lipoprotein resulted in additive accumulation of cholesteryl esters. Cholesteryl esters in cells loaded with triglyceride by oleate treatment were mobilized in response to dibutyryl-cAMP to an extent similar to that in cells containing low triglyceride concentrations. Dibutyryl-cAMP stimulated cholesteryl ester mobilization under all conditions, and stimulated triglyceride mobilization when adequate fatty acid acceptors were available. The results indicate that while triglyceride accumulation in MA-10 cells promoted cholesteryl ester deposition, it did not impair cAMP-dependent cholesteryl ester hydrolysis or steroid hormone production.


Antiviral nucleoside diphosphate diglycerides: improved synthesis and facilitated purification. Cytidine diphosphate diglyceride and its analogs have previously been synthesized by condensing phosphatidic acid with the monophosphomorpholidates of the various nucleosides. Yields have been low and purification of the product has been difficult. We report here an improved method for the synthesis of nucleoside diphosphate diglycerides with potential antiviral activity. Phosphatidic acid was activated with morpholine in the presence of dicyclohexylcarbodiimide to phosphatidic acid morpholidate. This compound was condensed with the 5'-monophosphate of the anti-HIV agents 3'-azido-3'-deoxythymidine, 3'-deoxythymidine or 2',3'-dideoxycytidine, and the monophosphate of the anti-HSV agent acyclovir. The resulting nucleoside diphosphate diglycerides are potential candidates for improved antiviral action when compared to the parent nucleoside analogs. Compared to the older method for the preparation of cytidine diphosphate diglyceride and analogs thereof, the new method has several advantages: reaction times are reduced from several days to several hours and the yield of the reactions is generally increased from 20-40% to between 50 and 80%. In addition, the purification of the compounds is greatly facilitated due to the small amount of phosphatidic acid remaining in the reaction mixture.


The use of magnetic resonance imaging in diagnosing Wilms' tumor mimicking a lumbosacral strain and sprain. Wilms' tumor is the most common tumor of the kidney in childhood. This article discusses the case of a young adult with a Wilms' tumor whose initial presentation appeared similar to a lumbosacral strain. The use of magnetic resonance imaging in diagnosing Wilms' tumor is discussed, along with the common clinical picture of the disease.


Origin of aldosterone in trypsin-stimulated rat adrenal zona glomerulosa incubations. The time-course for the in-vitro secretion of aldosterone and 18-hydroxycorticosterone (18-OH-B) by rat adrenal whole capsular tissue (largely zona glomerulosa) was studied under control and stimulated conditions. The stimulatory effect of trypsin was relatively delayed, and the steroids were significantly enhanced only after 1 h, in contrast to the actions of ACTH, which produced effects after 15 or 30 min. Tissue-sequestered 18-hydroxydeoxycorticosterone (t-18-OH-DOC), which is not affected by ACTH, was significantly depleted by trypsin, but secreted 18-OH-DOC was not consistently affected by either stimulant. In contrast to the apparent mobilization of t-18-OH-DOC, the conversion of exogenously added [3H]18-OH-DOC to [3H]18-OH-B was inhibited by trypsin, and aldosterone was unaffected. When trilostane was added to inhibit de-novo steroidogenesis, under conditions in which the steroid secretory response to ACTH is completely inhibited, aldosterone and 18-OH-B secretion was still stimulated by trypsin although yields were lower. Compared with controls, trilostane reduced t-18-OH-DOC concentrations, and trypsin caused a further depletion. In other studies, glomerulosa plasma membrane enriched preparations were homogenized and centrifuged, and the supernatants were dialysed and added to incubations of dispersed zona glomerulosa cells in the presence or absence of stimulators of aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)


Atrial natriuretic peptide is involved in the ACTH response to stress and glucocorticoid negative feedback in the rat. The role of atrial natriuretic peptide (ANP) in the ACTH response to stress and the glucocorticoid negative feedback control of ACTH release was investigated in adult male homozygous Brattleboro and adrenalectomized Wistar rats respectively, using the technique of immunoneutralization. The relatively low ACTH response to stress and the lack of arginine vasopressin make the homozygous Brattleboro rat a more rigorous and simpler preparation in which to test the hypothesis that ANP is involved in the ACTH response to stress. In both sets of experiments, blood sampling and injection of sheep anti-ANP or control serum were carried out in conscious animals through intra-atrial cannulae implanted 2 days previously under halothane anaesthesia. A 30-s exposure to ether resulted in a brisk twofold increase in the plasma ACTH concentrations in homozygous Brattleboro rats infused with anti-ANP, but not control serum. The injection of either dexamethasone, a potent glucocorticoid receptor agonist, or corticosterone resulted in a rapid and marked reduction in the plasma concentrations of ACTH in Wistar rats which had been adrenalectomized, under halothane anaesthesia, at least 21 days before experimentation. The inhibitory action of dexamethasone, but not corticosterone, was significantly reduced in animals infused with anti-ANP serum. These results show that the inhibition of ANP release into hypophysial portal blood is probably important for triggering the ACTH response to stress and that ANP may play a role in corticosteroid negative feedback control of ACTH release mediated by type II (glucocorticoid) receptors.


Evidence that acetylcholine is an inhibitory transmitter of heart interneurons in the leech. 1. In the leech, synaptic transmission between heart interneurons (HN cells) and between HN cells and heart motor neurons (HE cells) is blocked by bicuculline methiodide. 2. Gamma-aminobutyric acid, when applied focally onto the somata of HN cells or when added to the superfusate, has no effect on the membrane potential of HN cells. 3. Both acetylcholine (ACh) and the ACh agonist carbachol hyperpolarize HN cells and HE cells when applied focally onto their somata or into the neuropil or when added to the superfusate. 4. Inhibitory postsynaptic-potential-like responses elicited by focal application of carbachol onto the somata of HN cells and HE cells are blocked by bicuculline methiodide and are reversed when Cl- is injected into the cells. 5. Focal application of carbachol onto the somata of HN cells and HE cells increases membrane conductance. 6. The results indicate that HN cells use ACh as an inhibitory transmitter, that the postsynaptic receptors for ACh are blocked by bicuculline methiodide and that inhibition of HN cells and HE cells is mediated by an increased Cl- conductance.


Prevalence of vitamin B12 deficiency among geriatric outpatients. Healthy people can have low levels of cobalamin (vitamin B12) without symptoms or signs of cobalamin deficiency. Early detection of deficiency is imperative for treatment to be effective. Development of radioimmunoassay tests has greatly improved accurate determination of cobalamin (Cbl) levels. Nevertheless, results of studies of Cbl deficiency vary widely because of the variety of populations studied. In a prospective study, we tested 100 consecutive, unselected geriatric outpatients in a primary care setting to determine the prevalence of cobalamin deficiency. All patients, 65 years of age or older, who visited the office of one of the authors during a period of 11 consecutive working days, had their serum Cbl level checked. If the level was 299 pg/mL or lower, serum intrinsic factor and parietal cell antibodies, serum gastrin, part 1 Schilling test, serum methylmalonic acid, and total homocysteine were done, when possible, for the diagnosis of type A gastritis and intracellular Cbl deficiency. Sixteen percent of geriatric outpatients had serum Cbl levels of 200 pg/mL or below, and 21% had levels between 201 and 299 pg/mL. Among the 16 patients with levels < or = 200 pg/mL, 2 patients had macrocytic anemia, 3 patients had peripheral neuropathy, and 8 patients had type A gastritis. Among the 21 patients with levels of 201 to 299 pg/mL, 2 patients had peripheral neuropathy, 9 patients had type A gastritis, and none of the patients had macrocytic anemia. Among the patients whose methylmalonic acid and total homocysteine levels were determined, the results were high in 80% of those with Cbl levels < or = 200 pg/mL and in 33% of those with levels from 201 to 299 pg/mL. The prevalence of Cbl deficiency in geriatric outpatients was found to be higher than in any recent report. The lower limit of the normal range for Cbl level should be increased to 300 pg/mL.


Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen. Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.


Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes. Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.


Bovine polyomavirus, a cell-transforming virus with tumorigenic potential. The early region of bovine polyomavirus (BPyV) was tested for its cell transformation potential employing an assay of dense focus formation. Dense foci of morphologically transformed cells were observed upon transfection of primary rodent cells with a plasmid construct encoding the complete early region of BPyV under the transcriptional control of the long terminal repeat of Rous sarcoma virus. No transformation of primary rodent cells was observed upon transfection of these cells with a plasmid encoding the complete early region of BPyV under the control of its own transcriptional regulatory sequences. In BPyV-transformed cells, the viral sequences had become integrated into the cellular genome, and expression of large T antigen could be detected in a high percentage of cells. The transformed cells were demonstrated to be capable of anchorage-independent growth and to be oncogenic in immunocompromised newborn rats. Therefore BPyV should be considered as a potentially tumorigenic polyomavirus. Since many commercial batches of calf serum have been shown to be contaminated with BPyV, our observations may have implications for the use of calf serum in cell culture.


Nucleotide sequence of 21.8 kbp of variola major virus strain Harvey and comparison with vaccinia virus. A 21.8 kbp region of the genome of variola major virus (strain Harvey), a virus that caused haemorrhagic-type smallpox, has been sequenced and shown to possess 96% nucleotide identity to the corresponding region of vaccinia virus, the smallpox vaccine. Overall the gene arrangement in the two viruses is highly similar and individual open reading frames (ORFs) display a high degree of amino acid identity, for instance 26 of the 32 variola virus ORFs have > or = 90% identity with their vaccinia virus counterparts. A remarkable difference is the disruption of seven vaccinia virus ORFs into small fragments in variola virus. These include the variola virus homologue of vaccinia virus SalF2R, which encodes a protein related to C-type animal lectins, and SalF7L, which encodes an active 3 beta-hydroxysteroid dehydrogenase enzyme that contributes to vaccinia virus virulence. Upstream of the variola virus haemagglutinin gene there is a deletion of 1910 bp so that the equivalent of vaccinia virus gene SalF17R is truncated, and SalF16R, which shows amino acid similarity to the tumour necrosis factor receptor, is absent. The region sequenced includes the genes for thymidylate kinase and DNA ligase both of which are active in vaccinia virus and are highly conserved in variola virus. Other conserved ORFs with interesting homologies are those encoding profilin, superoxide dismutase and part of guanylate kinase. Two vaccinia virus genes encoding glycoproteins of the outer envelope of extracellular enveloped virus are also conserved in variola virus and this homology is likely to have contributed to the immunological protection which vaccinia virus evoked against smallpox. Lastly, there are multiple instances in which short oligonucleotide direct repeats flank a region absent from either variola or vaccinia virus.


Nucleotide sequence analysis of a unique near-terminal region of the tumorigenic poxvirus, Shope fibroma virus. Shope fibroma virus (SFV), a tumorigenic poxvirus, has a DNA genome of approximately 160 kb. Previous DNA sequence analysis of SFV has been mainly limited to the terminal inverted repetitions (about 12 kb at each end of the genome) and immediately adjacent regions. We have sequenced a 4 kb fragment located approximately 20 kb from the right-terminal hairpin. Within this region three complete and two partial open reading frames (ORFs) have been identified. Each of the putative polypeptides has sequence similarity to one or more previously identified poxvirus or cellular proteins, with homology to protein kinases, erythrocyte ankyrin and a vaccinia virus virulence-related protein (ORF N1L). The potential significance of these gene products with regard to the phenotype of SFV is discussed.


The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection. During an active infection with human cytomegalovirus (HCMV), viral antigen is consistently present in peripheral blood leukocytes. Two monoclonal antibodies (MAbs), CMV-C10 and CMV-C11, are commonly used in the HCMV antigenaemia assay to detect these cells in the peripheral blood of patients suspected of having an active HCMV infection. We demonstrate that the viral antigen detected by these MAbs is the viral structural protein pp65 and not an immediate early antigen as previously reported. Furthermore, significantly fewer leukocytes were found to be positive with MAbs specific for immediate early antigens.


Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins? A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.


Characterization and transcript mapping of a bovine herpesvirus type 1 gene encoding a polypeptide homologous to the herpes simplex virus type 1 major tegument proteins VP13/14. Using in vitro translation of hybrid-selected mRNA, we have previously shown that bovine herpesvirus type 1 HindIII fragment M encodes an abundant 94K polypeptide. Using immunoprecipitation and sequencing analyses, it has now been shown that the polypeptide is related to the major tegument protein VP8 and is homologous to the herpes simplex virus type 1 major tegument proteins VP13/14. The sequence of the VP8 gene (field isolate 34) is reported and compared to published data. Several differences between the sequences were detected, resulting particularly from base insertions/deletions generating three major frameshifts affecting an area of 87 amino acid residues of the encoded protein. In addition, sequence comparison revealed 29 single base alterations, excluding frameshift regions, producing 17 amino acid substitutions. Overall, 14.1% of the deduced amino acid sequences were divergent. We have also established that the last 152 nucleotides of the previously reported sequence correspond to the sequence of the minus not the sense strand. Finally, we report that the 4.4 kb transcript of the VP8 gene is initiated 39 nucleotides upstream from the translation start codon.


Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0. Dominant negative or trans-dominant mutants of viral proteins represent a new and exciting potential approach to antiviral therapy. Unfortunately, the extreme specificity of a given dominant negative mutant limits its general utility in treating a broad spectrum of viral diseases, since it can typically interfere with the activity of only a single viral polypeptide encoded by a single virus. However, it seems likely that dominant negative mutants of promiscuous viral trans-activator proteins, which by definition would repress rather than activate gene expression, should be able to inhibit infectious virus production for a number of different viruses. One such dominant negative mutant, derived from the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0, was found previously to behave as a powerful repressor of gene expression from an assortment of HSV-1 and non-HSV-1 promoters in transient expression assays. In the present study, this ICP0 mutant was found to be capable of inhibiting the replication of both HSV-1 and a completely unrelated virus, human immunodeficiency virus, in cell culture. The properties of this dominant negative mutant indicate that it may have potential as a means of treating diseases caused by a number of DNA and RNA viruses. Moreover, a truncated form of ICP0 which can hypothetically be created by alternative splicing was found to possess similar inhibitory capabilities, suggesting that a virus-encoded version of this dominant negative mutant may play a role in down-regulating HSV-1 gene expression during infection in vivo.


Nucleotide sequence analysis of genes encoding glycoproteins D and J in simian herpes B virus. The gene encoding glycoprotein D (gD) of simian herpes B virus (SHBV) was identified by hybridization with the gD gene of herpes simplex virus type 1 (HSV-1). The gene probe bound to a 2.6 kbp SalI-EcoRI fragment of SHBV DNA, which was cloned into a plasmid vector. The nucleotide sequence of the SHBV DNA fragment was determined. Two complete and one partial open reading frames (ORFs) were found. The nucleotide sequences of the two complete ORFs are 57% and 69% identical to HSV-1 genes US5 (encoding gJ) and US6 (encoding gD), respectively. The partial ORF showed 64% similarity with HSV-1 US7 (encoding gI). The SHBV gD gene revealed many features which are also found in the gD homologues of other herpesviruses. The positions of cysteine residues and receptor-binding sites for the predicted protein are shown to be highly conserved.


Adeno-associated virus type 2-mediated inhibition of human immunodeficiency virus type 1 (HIV-1) replication: involvement of p78rep/p68rep and the HIV-1 long terminal repeat. Microinjection of wild-type adeno-associated virus type 2 (AAV-2) DNA and infectious human immunodeficiency virus type 1 (HIV-1) proviral DNA into the nuclei of human epithelioid SW480 cells leads to specific inhibition of HIV-1 replication. Mutational analysis of the AAV genome showed that this negative interference can be assigned to a functional AAV-2 rep gene. Moreover, the p78rep/p68rep proteins are sufficient for the anti-HIV-1 effects. The rep gene also inhibits the expression of a chloramphenicol acetyl-transferase (CAT) gene driven by the U3/R portion of the HIV-1 long terminal repeat (LTR) in the absence of tat expression. This suggests that the U3/R portion of HIV-1 contains elements responsible for the AAV-2 rep-mediated inhibition of HIV-1 LTR-driven CAT gene expression and, probably, also of HIV-1 replication. The results add support for the general significance of AAV-2 and specifically the rep gene as tools for down-regulating heterologous gene expression.


Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated J. The host ranges of the Rous sarcoma virus (RSV) pseudotype RSV(HPRS-103) of a novel avian leukosis virus (ALV), strain HPRS-103, and representative RSV pseudotypes of subgroups A to F, have been determined in embryo fibroblasts from 12 avian species. Domestic fowl, red jungle fowl, Sonnerat's jungle fowl and turkey were susceptible to infection by RSV(HPRS-103); ring-necked pheasant, Japanese green pheasant, golden pheasant, Japanese quail, guinea-fowl, Peking duck, Muscovy duck and goose were resistant. The host range pattern of RSV(HPRS-103) differs from those of viruses of subgroups A to G and I, and provides support for placing the HPRS-103 strain of ALV in a new envelope subgroup, designated J.


The pathogenicity of two porcine rotaviruses differing in their in vitro growth characteristics and genes 4. The pathogenicity of two rotavirus variants, 4F and 4S, obtained following adaptation to cell culture of rotavirus from a diarrhoeic pig in China, was compared by serial passage in 24 gnotobiotic piglets. The rotavirus variants have markedly different growth characteristics in vitro, and their genome profiles differ only in the relative migration of genes 4. Both cell culture-grown variants replicated to an equal extent in gnotobiotic piglets and neither caused disease, although weight gain was slightly affected in piglets inoculated with the 4F variant. During five serial pig-to-pig passages, variant 4F became highly pathogenic at the fourth and fifth passages, causing severe diarrhoea and weight loss, and premature death in two animals. Piglets inoculated with rotavirus variant 4S remained healthy during all passages although weight gain was slightly affected. Mean duration and peak infectivity titres of virus shedding were similar for both variants. Thus, variant 4F, which grew slowly and produced small plaques in vitro and had the faster migrating gene 4, was pathogenic in pigs, whereas variant 4S was apathogenic.


Inhibition of rotavirus in vitro transcription by optimal concentrations of monoclonal antibodies specific for rotavirus VP6. Three monoclonal antibodies (MAbs) obtained from inoculation of mice with either a serotype 1 human rotavirus or rotavirus SA11 (serotype 3) inhibited the in vitro transcription of rotavirus SA11. Two of the MAbs exhibited a biphasic inhibitory response. Removal of antibody from MAb preparations by adsorption with Sepharose-Protein G reduced the inhibitory activity completely for all three MAb preparations. Analysis by radioimmunoprecipitation and Western blotting indicated that all three MAbs reacted with VP6. All MAbs also reacted with four group A rotavirus serotypes by ELISA, but did not cross-react with reovirus type 1, poliovirus type 2 or MA-104 cell lysates. Transcription of four rotavirus serotypes as well as epizootic diarrhoea of infant mice rotavirus was inhibited when tested with two of the MAbs. Transcription of both purified single-shelled virus and purified heat-activated double-shelled SA11 rotavirus was inhibited by purified MAb. Our results indicate that these MAbs can be used effectively to study the events associated with rotavirus transcription.


Sequence of genome segment 9 of bluetongue virus (serotype 1, South Africa) and expression analysis demonstrating that different forms of VP6 are derived from initiation of protein synthesis at two distinct sites. Bluetongue virus (BTV) VP6 is often resolved into two closely migrating bands by SDS-PAGE (VP6 and VP6a). RNA segment 9 of BTV-serotype 1 South Africa (encoding VP6) has been cloned as cDNA, and the complete sequence has been determined. Expression of this clone both in vitro and in tissue culture produced the same polypeptide doublet as seen previously in extracts from BTV-infected cells. Modification of the cDNA, including the removal of the first initiation codon, demonstrated that the two forms of VP6 are derived from initiation of protein synthesis at two distinct sites and not by post-translational modification.


Donated organ as a source of cytomegalovirus in orthotopic liver transplantation. The importance of the donated organ as a source of CMV was assessed in 120 patients following orthotopic liver transplant and the CMV infections that developed in these patients were graded by severity. Forty-four recipients were CMV antibody negative pre-transplant. Eighteen of these received organs from CMV antibody positive donors and 15 (83%) developed primary CMV infections, 13 (87%) of which were symptomatic. Twenty-six received organs from CMV antibody negative donors and only 2 (8%) became CMV positive post transplant (P less than 0.001). These data suggest that there would be a considerable advantage in matching CMV antibody negative recipients with negative donors. Forty-five percent of secondary infections were asymptomatic compared with 12% of primary infections, and only 11% became disseminated compared with 53% of primary infections. The secondary infections that followed transplantation of an organ from a CMV antibody positive donor were more likely to be symptomatic and were more severe than those in patients who received seronegative livers.


Accumulation and persistence of hepatitis A virus in mussels. Accumulation and persistence of hepatitis A virus (HAV) in the mussel Mytilus chilensis was evaluated. Under optimal filtration activity of mussels (temperature 12 degrees C, salinity 3%, feeding twice a day with Dunaliella marina), HAV was concentrated 100-fold from the surrounding water. Similar concentrations of HAV were reached in the filtration apparatus and in the digestive system (hepatopancreas). HAV persisted for about 7 days in mussels. Elimination of HAV from mussels was slower than elimination of poliovirus. Without feeding of mussels (causing low filtration activity), there was no measurable uptake of HAV into mussels, and depuration of HAV from mussels was slower. The ability of mussels to concentrate HAV was used successfully to monitor fecally contaminated river water for the presence of HAV.


Pre- and posttreatment serum antibody responses to HPV 16 E2 and HSV 2 ICP8 proteins in women with cervical carcinoma. Serum antibodies to early proteins of human papillomavirus type 16 (HPV 16 E2 protein) and herpes simplex virus type 2 (HSV 2 ICP8) can be measured by ELISA. In the serum of 122 newly diagnosed cervical carcinoma patients and age-matched controls, enhanced IgA antibody levels to an HPV-16 E2 protein derived peptide no. 245 indicated a 9.5-fold (95% confidence limits 2.8-57.2) relative risk of cervical carcinoma. No significant risk was found with a corresponding HPV 6 E2 peptide or HSV 2 ICP8. To evaluate the HPV 16 E2 peptide as a possible tumor marker for cervical carcinoma serial postoperative serum samples were tested from 27 women with cervical carcinoma. Antibody responses to the HPV 16 E2 peptide depended on the clinical stage. Stage I and II patients showed decreasing posttreatment IgA and/or IgG antipeptide antibody levels. Stage III and IV patients initially showed decreasing antipeptide antibody levels followed by increasing levels. These patients also showed increasing IgG antibody levels to the HSV 2 ICP8. However, increasing antibody levels to the HPV 16 E2 peptide indicated significantly (P less than 0.05) worse 2-year disease free survival (recurring disease) than did stable or decreasing antibody levels. The results suggest that serum antipeptide antibodies to the HPV 16 E2 peptide no. 245 can be used for the monitoring of cervical carcinoma.


Typing of human group A rotavirus with alkaline phosphatase-labeled oligonucleotide probes. Rotavirus (RV) in stools of children less than 1 year of age with diarrhea in Bangkok in 1989 were serotyped by monoclonal enzyme immunoassay (MEIA). RNA extracted from these specimens was tested for hybridization with alkaline phosphatase (AP) and 32P-labeled oligonucleotides constructed from the nucleotide sequences of VP7 of human G types 1 (HuG1Ac), 2 (HuG2Ac), 3 (HuG3Ac), and 4 (HuG4Ac). Of 148 specimens that contained RV, 72% (106/148) hybridized with RV G type specific AP-labeled oligonucleotides compared to 47% (70/148) that were serotyped by MEIA (P less than 0.001). Of 68 specimens that contained only one VP7 serotype (G-type), as identified by MEIA, 94% (16/17) of G1, 90% (27/30) of G2, 57% (4/7) of G3, and 36% (5/14) of G4 RV hybridized with the AP-labeled HuG1Ac, HuG2Ac, HuG3Ac, and HuG4Ac oligonucleotides, respectively. The probes for G1, 2, 3, and 4 RV were specific for each G type. The results of hybridizing specimens with 32P- and AP-labeled oligonucleotides were similar. After transcription and amplification of cDNA of gene 9, AP-labeled RV G type specific oligonucleotides hybridized with 90% (134/148) of RV specimens. The high sensitivity of these nonimmunological techniques could be of value in identifying G types of RV during vaccine trials.


Detection of HCV RNA in saliva, urine, seminal fluid, and ascites. Approximately half of the patients with type C hepatitis do not have a history of parenteral exposure. The route of nonparenteral infection remains unknown. To evaluate the possible role of body fluids, the existence of hepatitis C virus (HCV) RNA in saliva, urine, seminal fluid, and ascites was examined by "nested" polymerase chain reaction (PCR). Amplification of the HCV 5' noncoding sequences was carried out. The amplified product was confirmed by Southern blot hybridization and restriction endonuclease digestion. Among 34 patients with chronic liver disease who were positive for anti-HCV and serum HCV RNA, the prevalence of HCV RNA in body fluids was 100% (7/7) in ascites, 48% (15/31) in saliva, 24% (4/17) in seminal fluid, and 7% (2/29) in urine. The body fluids collected from 3 healthy subjects and 5 patients with chronic liver disease who were positive for anti-HCV but negative for serum HCV RNA were all negative for HCV RNA. Hence, the potential infectivity of body fluids in patients testing negative for serum HCV RNA can probably be discounted. Conversely, the presence of HCV RNA in saliva and seminal fluid of patients positive for serum HCV RNA suggests sexual and household contact as likely modes of nonparenteral transmission of type C hepatitis. Furthermore, the high prevalence of HCV RNA in ascites and saliva may have important implications in medical and dental practice.


Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays. A semiautomated dot blot assay and cDNA polymerase chain reaction (PCR) were used to study longitudinal anti-hepatitis C virus (HCV) recognition patterns in relation to presence of HCV-RNA in transfusion recipients and their infectious donors. In 9 recipients, 4 different patterns of HCV infection were observed: (A) persistent HCV carriage accompanied by chronic hepatitis in 6, (B) acute resolved hepatitis, but persistent HCV replication in one, and (C) continuous HCV replication without hepatitis in one and (D) acute resolved hepatitis with clearance of infection in one. This last self-limited infection was characterized by the disappearance of HCV-RNA as well as anti-HCV reactivity. In contrast, antibody reactivity persisted in 7 of 8 patients with chronic HCV infection who could be followed until 1990. Seven of the 9 recipients developed antibodies to all recombinant peptides in dot blot assay; one became positive for anti-C33 and anti-core and one developed anti-core only. The sequence of appearance of antibodies differed among individual patients. In 7 patients with full anti-HCV recognition patterns, the sequence of events was (mean and limits in days after transfusion): onset of hepatitis at day 50 (22-74), seroconversion of anti-C33 at day 91 (59-129), anti-core at day 133 (54-203), and anti-C100 at day 143 (59-365). The incorporation of C33 and core proteins, in addition to C100, in the second generation anti-HCV ELISA enhanced the detection rate in the HCV-infected transfusion recipients from 7/9 (78%) to 9/9 (100%).(ABSTRACT TRUNCATED AT 250 WORDS)


Treatment of chronic hepatitis C by continuous subcutaneous infusion of interferon-alpha. The effectiveness of a daily continuous infusion of interferon-alpha was evaluated in 12 patients (10 males, 2 females; mean age of 33 years, range 19-62) with biopsy-proven chronic active hepatitis C. Nine million units (MU) of recombinant interferon-alpha 2A (rIFN-alpha 2A) were administered by continuous subcutaneous infusion with a portable syringe pump, Graseby model MS 16A, for 24 h over 28 days. A significant decrease (P less than 0.01) in median serum alanine aminotransferase (ALT) levels was observed after the first week of treatment (96 IU/L, range 58-263) with respect to the pre-treatment values (188 IU/L, range 119-670). ALT became normal in four patients only by the fourth week. When IFN was interrupted, an increase in ALT was observed in all patients (1.5 to 5 times the pre-treatment values). The maximum decrease in ALT coincided with a significant increase in serum levels of the enzyme 2',5'-oligoadenylate (2-5A) synthetase (two to fourteen times the pretreatment values) and these parameters were inverse-correlated (r = -0.598, P less than 0.05). 2-5A synthetase levels returned to pre-treatment values after discontinuing IFN administration. Hepatitis C virus (HCV) RNA (as detected by the polymerase chain reaction using oligonucleotide primers of the NS5 region) was positive in all cases, remaining so during the treatment period. IgM antibody to HCV (as tested by ELISA) was present in 10/12 cases at baseline without changes throughout the study. No irreversible side effects were noted during therapy, which needed to modify the schedule.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of precursors of structural proteins VP1 and VP2 of hepatitis A virus. The morphogenetic pathway of hepatitis A virus (HAV), classified as a member of the enteroviruses within the Picornaviridae, still remains obscure and seems to differ considerably from that of poliovirus, the most studied representative of this genus. In order to elucidate the precursor/product relationship of HAV structural proteins, subviral particles, which represent more than 50% of the viral antigen produced in infected cells, were separated from mature virions and their polypeptide pattern was analyzed by polyacrylamide gel electrophoresis and immunoblotting using monospecific antisera. Whereas mature virions are composed of viral proteins VP1, VP2, and VP3, subviral particles contained VP0 and smaller polypeptides instead of VP2. Comparison of proteins of different strains of HAV showed that VP0 of strain HAS-15 migrated slower than that of strains MBB or GBM. During the course of the infectious cycle, VP0 accumulated and only small portions were converted to VP2 supporting earlier observations that encapsidation of RNA with concomitant cleavage of VP0 is rate-limiting, leaving a large amount of viral antigen in premature particles. Similar to VP0, accumulation of VP1 was observed and two immunologically related precursor proteins, p38 and p36, were found during the course of infection. Immunological characterization of p38 using antisera directed to the N-terminus of VP1 and to synthetic peptides located at the presumptive C- and N-termini of 2A suggests that p38 is VP1 delta 2A carrying 45 N-terminal amino acids of the P2-region.


Endonuclease analyses of DNA of human herpesvirus-6 isolated from blood before and after bone marrow transplantation. Three strains of human herpesvirus-6 (HHV-6) were isolated from peripheral blood mononuclear cells of a leukemic child with the antibody of HHV-6 before and after bone marrow transplantation (BMT); two strains were obtained before BMT and one after BMT. The DNA extracted from the three isolates was analyzed by six different restriction endonucleases. The cleavage profiles of two strains obtained before BMT were different, but the third strain isolated after BMT was identical with one of the two, which suggest reactivation of HHV-6 from the recipient's own body after BMT and possible mutation or super-infection of the virus in an immunocompromised patient.


Peripheral blood leukocytes of patients with Argentine hemorrhagic fever as effectors of antibody-dependent cell-cytotoxicity. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMNL) of patients with Argentine hemorrhagic fever (AHF) were tested as effectors (E) of antibody-dependent cell cytotoxicity (ADCC). 51Cr labeled chicken red blood cells (CRBC) coated with anti-CRBC or normal rabbit serum were used as targets (T). Three ADCC assays were performed with both effectors from patients: on admission (I), 4 days after the transfusion of immune plasma (II), and 30 days after the clinical onset (III). The ADCC values obtained displayed high variation between individuals. From the linear portions in the curves representing specific 51Cr release vs. E:T ratio plots, extrapolations were made to determine lytic units (LU), defined here as effector concentrations required to lyse 50% of the targets. The results were expressed as LU in 10(6) effector cells. The killing activity ranges of patients' PMNL (I = 1.04 +/- 0.34; II = 2.22 +/- 0.66; and III = 2.08 +/- 1.18) were not significantly different from that of 21 normal controls (1.19 +/- 0.36), except for range II (P less than 0.01). ADCC activity ranges of patients' PBMC (I = 3.40 +/- 1.06; II = 3.16 +/- 1.60; and III = 1.93 +/- 0.42) were not significantly different from that determined in 12 healthy subjects (1.86 +/- 0.40). These results demonstrate that patients' PBMC and PMNL can perform ADCC with efficiency comparable to normal effector cells, during the acute period of AHF, and in early convalescence. Consequently, ADCC can be a relevant mechanism in the clearance of Junin virus-infected cells.


Improved serodiagnosis of chronic hepatitis C in Japan by a second-generation enzyme-linked immunosorbent assay. To clarify the role of hepatitis C virus (HCV) infection in chronic liver disease, sera from Japanese patients which were negative by the original anti-HCV assay (Ortho) were subjected to a second-generation anti-HCV assay based on a combination of structural (C22) and nonstructural (C200) recombinant HCV proteins. Of 29 patients with chronic non-A, non-B hepatitis, 20 (69%) were anti-HCV-positive by the second-generation enzyme-linked immunosorbent assay (ELISA) and also positive by the reverse transcription-polymerase chain reaction (RT-PCR) which detects the HCV genome. Of 41 chronic hepatitis B virus carriers, 3 (7%) were positive by the second-generation ELISA; 1 of 3 was positive by RT-PCR. The HCV genome was detected in all cases positive for anti-HCV with high titers. Of 59 healthy subjects negative by the second-generation ELISA, none were positive by RT-PCR. These findings indicate that HCV is a major causative agent of chronic non-A, non-B hepatitis in Japan and that second-generation ELISA is specific and a more sensitive diagnostic assay for chronic hepatitis C.


Mechanism of agrin-induced acetylcholine receptor aggregation. Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.


Evidence that synaptic transmission between giant interneurons and identified thoracic interneurons in the cockroach is cholinergic. In the cockroach, a population of thoracic interneurons (TIs) receives direct inputs from a population of ventral giant interneurons (vGIs). Synaptic potentials in type-A TIs (TIAs) follow vGI action potentials with constant, short latencies at frequencies up to 200 Hz. These connections are important in the integration of directional wind information involved in determining an oriented escape response. The physiological and biochemical properties of these connections that underlie this decision-making process were examined. Injection of hyperpolarizing or depolarizing current into the postsynaptic TIAs resulted in alterations in the amplitude of the post-synaptic potential (PSP) appropriate for a chemical connection. In addition, bathing cells in zero-calcium, high-magnesium saline resulted in a gradual decrement of the PSP, and ultimately blocked synaptic transmission, reversibly. Single-cell choline acetyltransferase (ChAT) assays of vGI somata were performed. These assays indicated that the vGIs can synthesize acetylcholine. Furthermore, the pharmacological specificity of transmission at the vGI to TIA connections was similar to that previously reported for nicotinic, cholinergic synapses in insects, suggesting that the transmitter released by vGIs at these synapses is acetylcholine.


Biogenic amines modulate synaptic transmission between identified giant interneurons and thoracic interneurons in the escape system of the cockroach. In the escape system of the cockroach, Periplaneta americana, a population of uniquely identifiable thoracic interneurons (type A or TIAs) receive information about wind via chemical synapses from a population of ventral giant interneurons (vGIs). The TIAs are involved in the integration of sensory information necessary for orienting the animal during escape. It is likely that there are times in an animal's life when it is advantageous to modify the effectiveness of synaptic transmission between the vGIs and the TIAs. Given the central position of the TIAs in the escape system, this would greatly alter associated motor outputs. We tested the ability of octopamine, serotonin, and dopamine to modulate synaptic transmission between vGIs and TIAs. Both octopamine and dopamine significantly increased the amplitude of vGI-evoked excitatory postsynaptic potentials (EPSPs) in TIAs at 10(-4)-10(-2) M, and 10(-3) M, respectively. On the other hand, serotonin significantly decreased the vGI-evoked EPSPs in TIAs at 10(-4)-10(-3) M. These results indicate that octopamine, serotonin, and dopamine are capable of modulating the efficacy of transmission of important neural connections within this circuit.


Development of GABAergic connections in vitro: increasing efficacy of synaptic transmission is not accompanied by changes in miniature currents. Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous gamma-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and greater than 23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm2 to 15,600 cm2, indicating that a growing number of contacts is formed by a declining number of presynaptic neurons.


Synaptic plasticity in a regenerated crayfish phasic motoneuron. Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459-467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons.


Dispersal of adult females of Culex annulirostris in Griffith, New South Wales, Australia. The dispersal of Culex annulirostris, a major arbovirus vector in Australia, was studied in Griffith, N.S.W. using a mark-release-recapture technique. From an empirical model of dispersal, fitted to data on recaptured adults, the average distance dispersed was 6.8 km (95% c.l. 4.1-40.9 km), and 50% of the population dispersed 4.8 km or more. Maximum recorded dispersal was 8.7 km, and 2 individuals traveled more than 5 km in 1 day. The relevance of the findings to control strategy is discussed.


Differential phosphorylation of tau by cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II: metabolic and functional consequences. The effects of cyclic AMP-dependent protein kinase (cAMP-PK) or Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation on the binding of bovine tau to tubulin and calpain-mediated degradation of tau were studied. Both cAMP-PK and CaMKII readily phosphorylated tau and slowed the migration of tau on sodium dodecyl sulfate-containing polyacrylamide gels. However, cAMP-PK phosphorylated tau to a significantly greater extent than CaMKII (1.5 and 0.9 mol of 32P/mol of tau, respectively), and phosphorylation of tau by cAMP-PK resulted in a greater shift to a more acidic, less heterogeneous pattern on two-dimensional nonequilibrium pH gradient gels compared with CaMKII phosphorylation. Two-dimensional phosphopeptide maps indicate that cAMP-PK phosphorylates a site or sites on tau that are phosphorylated by CaMKII, as well as a unique site or sites that are not phosphorylated by CaMKII. Phosphorylation of tau by cAMP-PK significantly decreased tubulin binding and, as previously reported, also inhibited the calpain-induced degradation of tau. CaMKII phosphorylation of tau did not alter either of these parameters. These results suggest that the phosphorylation of site(s) on the tau molecule uniquely accessible to cAMP-PK contributed to the decreased tau-tubulin binding and increased resistance to calpain hydrolysis.


Quantification of rat cerebral cortex Na+,K(+)-ATPase: effect of age and potassium depletion. Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.


Iron-sulphur clusters with labile metal ions. A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd III from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [formula: see text] The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]0 cluster for divalent ions lie in the sequence Cd greater than Zn much greater than Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S]1+ as well as for [3Fe-4S]0. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-mu 2-sulphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least one additional ligand which may be from solvent (H2O or OH-) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn(II), that may trigger conformational change.


Existence of three subtypes of bradykinin B2 receptors in guinea pig. We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)


Functional roles of the ferritin receptors of human liver, hepatoma, lymphoid and erythroid cells. Ferritin receptors are present on the membranes of many normal and malignant cells. The binding specificity of these receptors for H and L subunits was examined using recombinant human ferritin homopolymers. At least two different types of ferritin receptors were found, one derived from normal rat, pig, and human liver which shows similar binding of H- and L-ferritin. The second receptor type, specific for the H-chain ferritin, has been identified on membranes of hepatic and other transformed cells, and of normal lymphoblasts and erythroid precursors. These two receptor types may have different metabolic functions: the hepatic receptor acting as a scavenger for circulating ferritin and possibly for iron exchange between hepatocytes and macrophages; the H-ferritin receptor having a regulatory role which is not directly related to iron metabolism. The expression of the H-ferritin receptor is closely related to the activation and proliferation state of the cells. Addition of H-ferritin to the culture medium of cells expressing the H-ferritin receptor resulted in inhibition of cell proliferation and of colony formation.


Growth factor-like effects mediated by muscarinic receptors in PC12M1 cells. Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.


Opioid-inhibited adenylyl cyclase in rat brain membranes: lack of correlation with high-affinity opioid receptor binding sites. Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.


Intracellular pH on protein kinase C and ionomycin potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP production in rat pinealocytes. In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.