[Current acquisitions in antiviral drugs (anti-HIV)]. These last years, numerous molecules have been developed to face HIV-1 infection. All viral replication steps are potential targets for new molecules. The most potent inhibitors of virus-cell adsorption are represented by the different sulfated, sulfonated and carboxylated polymers among which aurintricarboxylic acid (ATA). The soluble CD4 are also potent inhibitors of viral adsorption in vitro. Many compounds are active at the level of the reverse transcriptase (RT), particularly the 2',3'-dideoxynucleosides, represented by the three currently most used drugs in the clinic, AZT, ddC and ddI. The acyclic nucleoside phosphonates (PMEA, PMEDAP) have shown a broad spectrum activity against many human and animal retroviruses, and also unique pharmacological properties allowing infrequent administration. Finally, most recently, highly potent activity, without toxicity, has been demonstrated by TIBO, HEPT and other HIV-1 RT-specific inhibitors.


Characterization of [125I]acidic fibroblast growth factor binding to the cloned human fibroblast growth factor receptor, FGF-flg, on NIH 3T3 cell membranes: inhibitory effects of heparin, pertussis toxin and guanine nucleotides. The present studies were conducted to characterize the specific binding of recombinant human [125I]acidic fibroblast growth factor ([125]aFGF) to the cloned human fibroblast growth factor (FGF) receptor, flg, overexpressed on stably transfected NIH 3T3 mouse fibroblast (NFlg26) cell membranes. In the presence of 5 U/ml of heparin to block [125I]aFGF binding to membrane bound heparan sulfate proteoglycans, specific [125I]aFGF binding was optimal in the presence of 0.2 M NaCl and in a pH range of 7 to 9. [125I]aFGF labeled a single class of recognition sites with high affinity (Kd = 0.27 nM) and limited capacity (apparent maximum binding = 19.5 pmol/mg of protein). A similar estimate of ligand affinity (Kd = 0.25 nM) was determined from association and dissociation rate experiments. aFGF, basic fibroblast growth factor and several glycine-substituted point mutations of aFGF potently inhibited 0.1 nM [125I]aFGF binding. A variety of putative FGF receptor ligands including poly-L-lysines and poly-L-arginines, protamine, suramin and wheat germ agglutinin were shown to have weak or no affinity for the [125I]aFGF recognition site. Additional saturation studies, conducted in the presence of a lower (0.1 U/ml) heparin concentration, indicated that [125I] aFGF labeled both the high affinity (Kd = 0.02 nM) FGF-flg receptor and a separate class of lower affinity (Kd = 2 nM) recognition sites. Pretreatment of NFlg26 cell membranes with pertussis toxin resulted in a heparin-dependent decrease in the binding affinity (Kd values of 0.57-1.15 nM) of [125I]aFGF. Similar pretreatment with cholera toxin did not significantly affect [125I] aFGF binding. Guanine nucleotides were also found to significantly reduce 0.1 nM [125I]aFGF binding in a heparin-dependent fashion. The present data demonstrate that, in the presence of heparin, [125I]aFGF binds with high affinity to the cloned FGF-flg receptor on NFlg26 cell membranes. However, at a low heparin concentration (0.1 U/ml), [125I]aFGF binds to the FGF-flg receptor with higher affinity than was observed in the presence of 5 U/ml of heparin, and also binds a class of lower affinity recognition sites which are consistent with the labeling of cell surface heparan sulfate proteoglycans. The present data also indicate that agents which are known to interfere with receptor/G-protein coupling reduce the binding affinity of [125I]aFGF and suggest that the FGF-flg receptor may be coupled to a G-protein in addition to its intrinsic tyrosine kinase activity.


S-[2-(4-imidazolyl)ethyl]isothiourea, a highly specific and potent histamine H3 receptor agonist. The effects of a new agonist of histamine (HA) H3 receptors, Imetit (S-[2-(4-(imidazolyl)ethyl]isothiourea) were investigated in vitro and in vivo and compared to those of (R)-alpha-methylhistamine [(R)-alpha-MeHA], a prototypic drug. Imetit inhibited the binding of [3H](R-alpha-MeHA to rat brain membranes with a Ki value of 0.1 +/- 0.01 nM. The release of endogenously synthesized [3H]HA induced by K(+)-depolarization from rat brain slices and synaptosomes was inhibited by Imetit with EC50 values of 1.0 +/- 0.3 and 2.8 +/- 0.7 nM, respectively. Imetit behaved as a full agonist and was about 4 times more potent than (R)-alpha-MeHA and 60 times more potent than HA. Thioperamide, a selective H3 receptor antagonist, elicited a parallel rightward shift of the concentration-response curve for Imetit with an apparent Ki value of 5.6 +/- 1.4 nM. Imetit potencies relative to HA were less than 0.1% and only 0.6% at HA H1 and H2 receptor reference systems, respectively. Imetit was found not to be a substrate or an inhibitor of HMT. After p.o. administration to mice or rats, Imetit decreased (by approximately 50%) the tele-MeHA level in the cerebral cortex with ED50 values of 1.0 +/- 0.3 and 1.6 +/- 0.3 mg/kg, respectively. This effect was still maximal after 6 hr. The in vivo potency and duration of action of Imetit were in the same range as those of (R)-alpha-MeHA. It is therefore concluded that Imetit represents a new potent and selective HA H3 receptor agonist.


Imipramine's selective suppression of an L-type calcium channel in neurons of murine dorsal root ganglia involves G proteins. The whole cell recording technique was used to explore the depressant effect of the tricyclic antidepressant imipramine (IP) on calcium currents of neurons in cultures of murine dorsal root ganglia. The maximal whole cell current (ICa) mediated by the L-type calcium channel declined to 54% of control within 3 min of superfusing neurons with a solution containing 30 microM IP. In contrast, the T-type calcium current was unchanged. The IP-induced reduction of ICa was not associated with a change of the current-voltage relations of ICa. The depressant effect of IP on ICa was greatly reduced if neurons were pretreated with pertussis toxin or dialyzed with an intracellular solution containing guanosine 5'-O-2-thiodiphosphate. In contrast, superfusion of neurons with 5 mM 8-bromo-cyclic-AMP did not alter the effect of IP upon ICa. These data suggest that the selective suppressant effect of IP on the L-type calcium channel involves either an interaction with that region of the channel complex coupled to guanosine nucleotide-binding proteins or with guanosine nucleotide-binding proteins themselves.


Activation and desensitization of embryonic-like receptor channels in mouse muscle by acetylcholine concentration steps. 1. Pulses of acetylcholine (ACh) in concentrations between 0.1 and 1000 microM were applied repetitively to outside-out patches of enzymatically denervated (14 days) mouse muscle with the liquid filament switch. Solutions superfusing the patch could be changed rapidly (within 0.2 ms). 2. Single-channel activity was studied under steady-state conditions in the outside-out and in the cell-attached mode. The single-channel conductance was 26 pS in outside-out patches, characteristic for embryonic-like channels. Apparent mean open time was about 2.5 ms, a shorter component of closed times was 800 microseconds and burst length was about 5 ms. 3. Channel currents elicited by pulses of ACh were averaged. The time-to-peak current was concentration dependent and decreased from a level of about 10 ms below 10 microM to about 400 microseconds at 100 microM-ACh. 4. For a typical experiment, the average peak current, imax, increased from -0.4 pA with 0.1 microM to -82 pA with 1000 microM-ACh, close to the value at saturation. The half-maximal response was at 60 microM-ACh. The dose-response curves for imax had double-logarithmic slopes of 1.1-1.3, consistent with two binding sites at the embryonic nicotinic acetylcholine receptor (nAChR). 5. The current elicited by ACh pulses decreased rapidly after the peak. The time constant of desensitization increased from 20-50 ms with 1000 microM-ACh to up to more than a second with 1 microM-ACh. 6. The current in steady state (fully desensitized) increased up to 10 microM-ACh, but decreased slightly to values of imax/100 to imax/500 when higher concentrations were applied. 7. In addition to the well-known differences between adult and embryonic nAChR concerning the apparent mean open time and burst length, we found differences in the slope of the dose-response curve for imax, in the ratio of peak to steady-state response, and in the rise time of the response.


Characterization of membrane currents in single smooth muscle cells from the guinea-pig gastric antrum. 1. Smooth muscle cells, enzymatically isolated from the antrum of the guinea-pig stomach, were voltage clamped at room temperature using the whole-cell patch clamp technique. In physiological salt solution (PSS), step depolarization from a holding potential of -90 mV elicited inward calcium current (ICa) followed and superimposed by outward potassium current. 2. Outward current was divided into components depending on the presence of extracellular Ca2+ and others which were not activated as a result of Ca2+ influx. Ca(2+)-dependent components were (1) a fast transient component most likely representing Ca(2+)-activated K+ current (IK(Ca)) immediately triggered by the initial peak of ICa and (2) spontaneous transient outward currents (STOCs) apparently reflecting synchronized opening of IK(Ca) channels by cyclic release of Ca2+ from intracellular stores. Ca2+ influx-independent outward current could be divided into two main components: (1) a transient component (I(to)) showing voltage-dependent activation and inactivation and (2) a non-inactivating component (Ini). 3. I(to) activated with a threshold around -30 mV, was fully available at -90 mV and completely inactivated at -10 mV. The time course of both activation and inactivation of I(to) at different potentials could be described by single exponential functions. Time constants of activation decreased from 35 ms at -10 mV to 10 ms at +40 mV. The time constant of inactivation was about 2 s and only weakly voltage dependent. Time constants for exponentially developing recovery from inactivation of I(to) ranged from 0.1 s at -100 mV to 10 s at -30 mV. I(to) was insensitive to 4-aminopyridine (4-AP, 5 mmol/l), slightly sensitive to tetraethylammonium (TEA, 10 mmol/l), but substantially inhibited by caffeine (10 mmol/l) and Cd2+ (5 mmol/l). Estimates of the single-channel conductance by current fluctuation analysis indicated a small value of about 2.5 pS. 4. The action of TEA on current elicited from a holding potential of -10 mV indicated a major contribution to Ini of a distinct component (Ini,K) that was completely blocked by this substance at a concentration of 10 mmol/l. Ini was almost unaffected by 4-AP (5 mmol/l) and caffeine (10 mmol/l), but strongly suppressed by Cd2+ (5 mmol/l). Current fluctuation analysis of Ini,K gave a value for the single-channel conductance of about 60 pS. 5. Ca2+ inward current was studied in PSS ([Ca2+]o = 2.5 mmol/l) using pipette solution in which K+ was replaced by Cs+ to suppress outward K+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)


Distribution of adhesion receptors in recurrent oral ulcers. When activated under physiologic or pathologic conditions leukocytes adhere to one another or to other cell types. Adhesion receptors mediate these interactions. In the study reported here, the distribution of the adhesion receptors LFA-1 (CD11a/CD18), ICAM-1 (CD54), CD2 and LFA-3 (CD58) in recurrent oral ulcers (ROU) were studied. Nine tissue specimens from five female patients (mean age 33 yr, age range 21-40 yr) with ROU were studied using the avidin-biotin-peroxidase complex (ABC) method. The main mononuclear cell infiltrations were in lamina propria (LP) and the epithelium next to the basement membrane (BM), laterally to the ulcers. In this area, ICAM-1 was strongly expressed in capillaries and in postcapillary venules. LFA-1, LFA-3 and CD2 were expressed in 65 +/- 1.1%, 70 +/- 16% and 80 +/- 1%, respectively, of all mononuclear cells. The findings indicate that LFA-1/ICAM-1 and CD2/LFA-3 interactions may play roles in cell to cell adhesion events in ROU.


Immunoreaction of keratin, actin, S-100 protein and rat-EGF in duct-ligated rat salivary glands. Duct-ligated submandibular and sublingual glands of rats were evaluated immunohistochemically for changes in keratin (MoAb 1164), actin, S-100 protein and rat-EGF (rEGF). Normal salivary glands were reactive for keratin, S-100 protein and rEGF in the granular convoluted tubule (GCT) and duct cells, and for actin in the myoepithelium. Submandibular glands showed a marked reduction of S-100 protein and rEGF staining following duct ligation, and no increased staining of proliferating epithelial cells of the late stage in duct ligated glands. Sublingual glands revealed no marked changes for actin staining in myoepithelial cells, irrespective of atrophic changes occurring in acinar and duct cells after duct ligation. Immunohistochemical patterns differed for each type of gland; changes associated with the obstructive lesion were more prominent in the submandibular gland.


Expression of collagenase and potential transcriptional factors c-fos and egr-1 in periodontal gingival fibroblasts. In view of the important role of fibroblast-type collagenase in the pathogenesis of periodontal disease (PD), we investigated the expression of this metalloproteinase in primary cultures of non-stimulated fibroblasts dissected from gingival tissues of patients with generalized moderate and localized severe chronic adult PD. Enhanced hybridization signals for collagenase RNA were observed in 8/8 PD-cases when compared with equivalent RNA amounts extracted from normal fibroblasts. Since both the proto-oncogene c-fos and the "early growth response" gene egr-1 might be involved in the transcriptional regulation of the collagenase gene expression in vivo, we also compared the relative expression of both potential transcriptional factors with collagenase RNA in the same fibroblast cytoplasmic extracts. Hybridization signals indicated elevated RNA amounts for c-fos in 8/8 PD-cases and for egr-1 in 7/8 PD-cases when compared with the cells from non-inflamed tissue. In periodontitis gingival tissue specimens, immunolocalization of collagenase could be confirmed in fibroblasts, macrophages and epithelial cells in situ. Collagenase label was not widely distributed within the tissues, but concentrated at the interface between epithelium and connective tissue. The data provide the first evidence that gingival fibroblasts producing elevated levels of collagenase RNA amounts express also c-fos and egr-1 indicating a crucial role for both genes in cellular proliferation and collagenase expression in gingival and periodontal tissue destruction in vivo.


Cytokeratin profiles in dyskeratosis congenita: an immunocytochemical investigation of lingual hyperkeratosis. In dyskeratosis congenita, the hyperkeratotic lesions affecting the mucous membranes have a propensity to undergo malignant change. Unfortunately there is presently no reliable method for predicting such an outcome. Oral mucosal biopsies were obtained from a case of dyskeratosis congenita and keratin expression identified using a panel of antikeratin antibodies. Marked changes were found from keratin profiles established for normal oral mucosa. In particular coexpression of three separate type 1 keratins (K16, K10 and K13) were observed in the ventral tongue lesion. The keratin pattern found in the tissue biopsies examined is suggestive of an unusually immature or disturbed state of tissue differentiation, and as such may be indicative of future malignant change.


Involucrin expression in some oral lichenoid lesions. The aim of this investigation was to assess the reproducibility of a study which reported that involucrin could be used as a diagnostic marker in oral lichenoid lesions. Twenty-eight formalin-fixed, paraffin-embedded specimens were examined. They included tissue from 13 patients with lichen planus, 11 with drug-related lichenoid reactions, one with dysplasia in a lichenoid lesion and three with normal oral mucosa. In each case the biopsy site was the buccal mucosa, PAS stained smears for fungi were negative and immunofluorescence results supported the diagnosis. An immunoperoxidase technique was used to demonstrate involucrin reactivity. In all sections there was consistent positive staining for involucrin in the superficial layers of the epithelium and all the basal layers were negative. In conclusion there were no demonstrable differences in the involucrin reactivity between the different lesions.


Direct immunofluorescence of oral mucosal biopsies: a comparison of fresh-frozen tissue and formalin-fixed, paraffin-embedded tissue. The diagnosis of some oral mucosal diseases can be facilitated by the use of direct immunofluorescence. Fresh frozen tissue is preferred when using this technique, however frequently the material received by the histopathology laboratory has been fixed in formalin. Enzyme digestion of tissue sections glued to glass slides prior to incubation with antibodies was shown in this study to give comparable results with direct immunofluorescence on fresh frozen tissue and can be used for diagnostic purposes when fresh frozen tissue is unavailable or not representative of the disease process.


Immunohistochemical and electronmicroscopic studies of obstructive lesions in submandibular glands. Obstructive sialoadenitis was examined by immunohistochemical techniques for keratin (MoAb KL1, PKK1 and K8.12) and actin. Electronmicroscopy (EMS) was used to identify ultrastructural changes in myoepithelial cells and ductal basal cells. With immunohistochemistry, actin staining was used as a marker of myoepithelium, MoAbs KL1 and PKK1 for ductal luminal cells, and MoAb K8.12 for ductal basal cells. Histologic features of the lesion usually showed degenerative changes of acinar and duct cells with cell infiltration and fibrous replacement. Immunohistochemical findings indicated that actin staining in the changed myoepithelial cells was irregularly positive or negative, and also keratin staining in luminal and ductal basal cells was reduced or disappeared. Ultra-structural features of the changed myoepithelial cells indicated that these cells appeared less altered than adjacent acinar and ductal cells and showed increased amounts of lipid droplets and lipofuscin granules, and also wrinkled processes filled the prominent myofilament material.


Three-dimensional representations of lingual cortical defects (Stafne's) using silicone impressions. Lingual cortical defects of the mandible are common radiographic entities found predominantly in adult males. Due to the benign nature of these defects and infrequent need for exploratory surgery, few paleopathologists and clinicians have had the opportunity to examine their contents. The present study, using silicone impressions, provides three-dimensional representations reflecting the size and shape of soft-tissue masses once filling 10 unusually large lingual defects. The 10 specimens were chosen from an international sample of 6,330 dry mandibles, 115 of which had lingual cortical defects.


Positively cooperative cAMP phosphodiesterase attenuates cellular cAMP responses. We have shown that growth of S49 WT mouse lymphoma cells for 24 hr in 3 nM epinephrine produced very significant desensitization of subsequent cellular cAMP responses to challenges with higher concentrations of epinephrine. The effects of this long-term treatment (LTT) were obvious in intact cells and also when adenylate cyclase activity was measured in semi-purified membranes. Beta 2-adrenergic receptors (beta 2AR) were decreased by LTT, and the desensitization of adenylate cyclase was due at least in part to this down-regulation. When mouse L cells transfected with WT beta 2AR from hamster lung (L-WT beta 2AR cells) were subjected to LTT, the attenuation of adenylate cyclase in membranes was obvious, but the consequences of LTT on intact L-WT beta 2AR cells were highly equivocal. That is, when the effects of epinephrine on cellular cAMP levels were measured in LTT or control L-WT beta 2AR cells little desensitization was apparent. Further, cellular cAMP excursions in response to even very high concentrations of epinephrine were very small in control L-WT beta 2AR cells as compared to control S49 WT cells. Subsequent experiments have shown that L-WT beta 2AR cells possess a phosphodiesterase (PDE) which demonstrates marked positive cooperativity with cAMP with a Hill coefficient of 2. The EC50 for cAMP hydrolysis was approximately 30 nM in cell free preparations. cGMP was a positive allosteric effector of the L-WT beta 2AR cell PDE. Further, when cellular cAMP levels in intact L-WT beta 2AR cells were raised above a threshold by treatment with 0.5 microM forskolin and 2 mM IBMX with the epinephrine challenge, the effect of LLT became obvious in the intact cell system. These data demonstrate that cAMP responses to hormones are greatly decreased in systems where the predominant PDE demonstrates positive cooperativity for cAMP.


Guidelines for a scientific presentation. Guidelines for a projected scientific presentation can assist the speaker in preparing an effective audiovisual presentation. This article highlights the factors that improve the quality of an audiovisually assisted presentation and enhance successful communication.


Sutured laparoscopic cholecystojejunostomy evolved in an animal model. In a series of acute and chronic experiments conducted in young pigs during a 2-year period, we have evolved a quick knotting technique to start and end a suture line and developed a curved 'Endoski' needle to improve needle passage through the tissues. These have been employed in the construction of cholecystojejunal anastomoses using a semi-closed technique with a single layer (deep seromuscular) of continuous polyglactin sutures. In chronic experiments in pigs, patent well healed bilioenteric anastomoses, which conveyed bile satisfactorily after ligation of the common bile duct, distal to the cystic duct, were achieved. A virtual absence of adhesions in these animals was noted.


Paediatric trauma in northern Iraq: the Kurdish refugee crisis. In May 1991 a team of five doctors and two nurses from Edinburgh hospitals were flown to Northern Iraq to assist in the Kurdish refugee relief effort. The two surgical members of the team helped to re-establish surgical services in a small hospital in the Iraqi town of Zakho. During an 8-day stay in the hospital, frequently in extremely difficult circumstances, the surgical team undertook 19 major and 15 minor surgical procedures in 25 patients; 11 major and eight minor procedures were undertaken in 15 children under the age of 16 years. The largest group of children treated had suffered bomb-blast injuries from unexploded ordinance; injuries sustained were primarily to the hands, face and upper trunk. Road traffic accidents and burns were also common. Two children requiring postoperative intensive care were evacuated by military helicopter at night to hospital facilities in Turkey.


Transhiatal oesophagectomy in the management of advanced oesophageal carcinoma. Thirty-seven patients have undergone transhiatal oesophagectomy for tumours in the upper (n = 3), middle (n = 12) and lower (n = 22) thirds of the oesophagus. Four tumours arose in association with Barrett's oesophagus. Dysphagia for solids was the presenting symptom in 95% of cases. Orringer's technique was used and all cervical anastomoses were hand-sewn. The median duration of surgery was 2.9 (range 1.5-4.0) h and the 30-day hospital mortality rate was 16% (six patients). Respiratory complications were considerable (48%) and accounted for three deaths. Median postoperative stay was 21 (range 13-53 days) while median stay in the intensive care unit was 8.5 days. The majority (97%) of patients had stage III disease and 14 (38%) had lymph node involvement. The actuarial survival was 56% at 1 year and 31% at 2 years. Of the operative survivors, 90% resumed normal swallowing although 17 (55%) required outpatient dilatation. Transhiatal oesophagectomy provides safe and efficient palliation while mortality and 1-year survival rates compare with the transthoracic approach.


Hepatic hydatid cysts: presentation and surgical management in Yemen. A total of 97 patients with hepatic hydatid cysts were examined and surgically treated over a period of 4 years. All patients, most of whom (86; 89%) were female, presented with upper abdominal swellings. Ultrasonography was of great value in confirming the diagnosis in all cases. All the hydatid cysts were treated with minimal surgical interference and the residual cavities were drained after surgery. Formalin (0.5%), the only scolicidal agent available in Yemen, was used in 73 cases of the series. There were no deaths and no recurrences.


Management of perianal sepsis in a district general hospital. Perianal sepsis remains a common surgical problem. A total of 121 patients undergoing surgery for perianal abscess and/or fistulae over a 2-year period was studied. Of these, 50 patients (41.3%) had suffered from previous perianal sepsis (not necessarily resulting in hospital attendance). Ninety-one patients underwent incision and drainage of abscesses (ischiorectal and perianal) for the first time, whereas eight patients underwent drainage of recurrent abscesses. Fistulae were identified when the abscess was drained in 14 of 91 patients, and a further ten patients subsequently developed fistulae. Twenty-two patients presented with a discharging fistula. A high yield of bowel organisms was present in patients with coexisting fistulae (88%), recurrent abscesses (75%) and in those who subsequently developed fistulae (83%). We confirm that such a growth can be used to identify patients who will benefit from further examinations.


Colorectal surgeons in district general hospitals produce similar survival outcomes to their teaching hospital colleagues: review of 5-year survivals in Manchester. A study of 578 patients treated for colorectal cancer in the North-west region comparing survival after surgery in teaching and non-teaching hospitals was performed. All patients had a minimum of 5 years follow-up. A greater proportion of elderly and emergency patients were treated in the non-teaching hospitals. The number of operative mortalities and 5-year survival figures for all causes of death and for colorectal deaths alone were similar in teaching and non-teaching hospital patients.


C-reactive protein in the differential diagnosis of the acute abdomen, especially acute appendicitis. Serum C-reactive protein (CRP) was qualitatively determined before surgery in 189 patients undergoing appendicectomy. The sensitivity, specificity, negative predictive value and overall accuracy of CRP were assessed: CRP had a high specificity (76.3%) but a sensitivity of only 39.7%. CRP may be of value in the assessment of acute abdominal pain, by helping to exclude from surgery those patients with normal appendices.


Purification and partial characterization of immobilization antigens from Ichthyophthirius multifiliis. Ichthyophthirius multifiliis, a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elicited immobilizing antibodies (Ab) when injected into rabbits. An effort was made to purify and characterize these Ag (referred to as immobilization Ag) because of their potential role in protective immunity in fishes. Mice immunized with theront cilia were used for production of immobilizing monoclonal antibodies (MAb). Hybridomas were screened by indirect immunofluorescent light microscopy and immobilization of live parasites. Six hybridomas producing immobilizing MAb were cloned. Immobilizing MAb were used to affinity purify Ag solubilized with Triton X-114 and Na deoxycholate. Two membrane protein Ag of approximately 48 and 60 kDa were identified. Immobilizing MAb failed to react with these Ag on Western blots and, conversely, MAb that reacted with the Ag on Western blots did not immobilize live organisms. These results suggest that immobilization required native conformational epitopes which were altered by Western blotting procedures. Individual MAb reactive on Western blots recognized both the 48- and 60-kDa proteins indicating the presence of common epitopes. Affinity purified Ag elicited immobilizing antisera when injected into rabbits, mice, and channel catfish.


Quality and interpretation of operative cholangiography in a district general hospital. The quality and interpretation of operative cholangiography were assessed in 128 patients undergoing cholecystectomy. The quality of each cholangiogram was assessed by calculating a cholangiogram score according to the anatomical structure visualized. Out of a maximum possible score of 5, 26% of cholangiograms achieved a score of less than or equal to 3 and were considered as technical failures. The sensitivity, specificity and negative predictive values regarding the surgeon's interpretation of the cholangiograms were high and compared well with the radiologist's assessment. However, the positive predictive value for the surgeon's assessment of the cholangiograms was 74% compared with 95% for the radiologist (P less than 0.02). Improvement in cholangiography can only be achieved by greater attention to detail and perseverance. After an adequate examination, the only criteria for common bile duct exploration should be the presence of filling defect(s).


A drip is unnecessary after cholecystectomy. A randomized controlled study of 93 patients undergoing a cholecystectomy was performed to examine the need for intravenous fluids in the postoperative period. Forty-five patients were randomized to have only oral fluid, and these patients suffered significantly less in the way of haemodilution as measured by changes in packed cell volume (P = 0.0001), haemoglobin (P = 0.0001) and urea (P = 0.036) than those who routinely received intravenous fluids. Although questionnaires at the time of discharge showed that patients themselves did not object to an intravenous infusion, recent studies linking deep vein thromboses to haemodilution, coupled with the findings presented here, provide an argument against their routine and often unconsidered use.


Colorectal cancer in South Tyneside: a personal audit. A detailed retrospective audit over a 10-year period of 162 patients presenting with colorectal cancer to one surgeon without a specific interest in colorectal surgery was undertaken. The modes of presentation and potential sources of delay in treatment were analysed. The 30-day hospital mortality rate was 10.1% and most of these patients had incurable disease. The crude survival 1 year after the study was 34% (mean follow-up 6 years); at 3 years after study, it was the same (8 years mean follow-up). Survival data are also given in relation to the presentation, and operative and histological findings. Preoperative radiotherapy for rectal cancers is discussed and assessed. During the last 3 years, 49 further patients have presented. The operative mortality rate was 8%. It is suggested that improved surgical treatment, particularly in the acute case, has been responsible for the reduced mortality rate. Only 51% of patients underwent operation with a reasonable prospect of cure. It is suggested that no improvement in the prospects of patients with colorectal cancer will occur until pre-symptom diagnosis from screening techniques is more commonly available.


Partial weight bearing after operations for hip fractures in elderly patients. Factors affecting a patient's ability to carry out partial weight bearing after operation for hip fracture were studied in 100 patients. Seventy-six were able to do so. Significant factors included the muscle power of the good limbs and the mental state, whereas age, body-weight and type of operation were not significant. Logistical regression analysis showed that it was possible to predict a patient's partial weight bearing potential by simply testing the left hand grip and the 'Set' test score.


Treatment of undisplaced subcapital fractures. A total of 157 patients with undisplaced subcapital fractures was studied with reference to outcome depending on the method of treatment; 135 were treated surgically and 22 were treated conservatively. The main complication of treatment was non-union, with an overall incidence of 5.7%. Conservative treatment resulted in an incidence of non-union which was approximately twice that of patients who were treated surgically. Patients treated surgically, however, had significant postoperative complications in 14.8% of cases. Although conservative treatment has an increased incidence of non-union, it is a safe method of treatment and we feel it still has a place in selected patients.


Trends in infection prophylaxis in orthopaedics. The current measures employed by consultant orthopaedic surgeons in Scotland to prevent infection were established by postal questionnaire. Our findings were compared with those of a similar study carried out 5 years previously. An increasing number of surgeons use routine systemic antibiotic prophylaxis for total hip arthroplasty (99% versus 91% 5 years ago), in treating compound fractures (89% versus 75% 5 years ago) and for internal fixation of closed fractures with metal implants (49% versus 12% 5 years ago). Cephalosporins are increasingly used as the antibiotic of choice. By starting antibiotics earlier than the day of surgery or continuing for more than 24 h after surgery, just over half the surgeons questioned administer antibiotics for longer than would seem to be necessary for elective surgery.


The beneficial effect of dextran on anastomotic patency and flap survival in a strongly thrombogenic model. Dextran lowers the probability of thrombus formation, by reducing platelet aggregation and adhesion and by increasing fibrinolysis. All studies to date using dextran for microvascular reconstruction have examined only short-term (1 to 2 hr) patency in isolated vessels. The current study used an established thrombotic model (inverted sleeve interposition graft), to investigate the effect of dextran on the long-term survival of pedicle flaps. A6- x 3-cm epigastric flap was elevated. A 2-mm inverted sleeve interposition graft was placed on the artery side of the pedicle by microvascular techniques. One group received dextran (17 cc/kg) 2 hr preoperatively and every 6 hr postoperatively for the next 72 hr. A control group received saline on the same schedule. Survival was assessed on postoperative day 7, at which time necrosis was obvious. Several parameters (clotting studies and electron microscopy) were studied to characterize the phenomenon more clearly. Only 25 percent of saline-treated flaps survived (2/8), while all dextran-treated flaps survived (7/7) (p less than 0.02, Fisher exact test). Thus, dextran allowed flaps to survive by preventing thrombus formation, despite a strong thrombogenic focus.


Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. When western blots of human sperm proteins solubilized by acid extraction (presumably mainly acrosomal proteins) or by sodium dodecyl sulfate (SDS) were probed with biotin-conjugated Pisum sativum agglutinin (PSA), distinct sets of proteins were labelled in both preparations. When smears of human spermatozoa were treated with methanol either for 30 s or for 15 min and then exposed to FITC-conjugated PSA, the resulting fluorescence pattern essentially depended on the time of methanol treatment. With the longer treatment, fewer spermatozoa showed selective acrosomal labelling and more were labelled uniformly throughout, without a clear predilection for a single sperm region. With the shorter time of methanol treatment, the poorly topographically differentiated, whole-cell labelling was typical of dead spermatozoa as confirmed by a close correlation between the percentages of spermatozoa showing this type of labelling and of those stained supravitally with Hoechst 33258. The preferential whole-cell labelling of dead spermatozoa with PSA is considered to be due to increased availability of the nonacrosomal set of PSA-reactive sites in dead spermatozoa after a short treatment with methanol, whereas this treatment is probably not sufficient to expose most of these sites when applied to living spermatozoa. The simplicity of the staining protocol makes this method feasible in routine work in a number of clinical and research applications.


Genes encoding the alpha and beta chains of follicle-stimulating hormone are not sites for the Booroola (FecB) mutation in sheep. Bovine cDNA probes for the beta-subunit of follicle-stimulating hormone beta (FSH beta) and the alpha-subunit of the glycoprotein hormones identify genetic variation (polymorphic restriction fragments) near these genes in sheep. The inheritance of the polymorphic restriction fragments was studied in half-sibling pedigrees generated by mating heterozygous (B+) rams to non-carrier (++) ewes so that the co-inheritance or genetic linkage to the Booroola (FecB) locus and the alpha- and beta-subunits of FSH could be analysed. Genetic recombination was observed between the FSH beta locus and the FecB locus in all five families studied and between the alpha-subunit and the FecB locus in the two families studied. We conclude that the FecB mutation does not lie within the FSH beta- or alpha-subunit genes encoding the heterodimeric hormone FSH, and that the high concentrations of FSH observed in carrier ewes must result from indirect actions of the FecB mutation on the synthesis, processing, storage, release or metabolism of FSH.


Spontaneous reflex sympathetic dystrophy (Sudeck's atrophy) syndrome associated with idiopathic thrombocytopenia. A 37-year-old woman developed reflex sympathetic dystrophy (RSD) (Sudeck's atrophy) syndrome in the right foot. Simultaneously, she had idiopathic thrombocytopenia. There was no history of injury or bone fracture before onset of RSD. Spontaneous RSD associated with idiopathic thrombocytopenia has rarely been described. A possible relationship between the 2 conditions is discussed.


Innervation of bone from healthy and arthritic rats by substance P and calcitonin gene related peptide containing sensory fibers. Mechanical stress causes remodelling of bone, a transformation of bone structure by physical forces through an unknown mechanism. Inflammation also affects bone structure, through altered use and the production of various inflammatory mediators. The peripheral nervous system may play both a sensory and an efferent role in the mechanical and inflammatory influences on bone structure. We studied the occurrence of substance P and calcitonin gene related peptide (CGRP) containing nerves in periosteal tissue, bone marrow, diaphysis and epiphysis of the ankle and knee joints of healthy and adjuvant arthritic rats. In arthritic animals, only ankle joints were affected by the inflammation. The periosteum was richly innervated both in healthy and arthritic animals. In arthritic rats few nerve fibers penetrated the woven, callous bone underlying the periosteum. Also bone marrow contained substance P and CGRP immunoreactive nerves in normal bone, whereas the hypercellular bone marrow of arthritic rats showed a decrease in the density of substance P and CGRP containing fibers. Epiphysis had a dense innervation compared to diaphysis. In contrast to large erosions, small peripheral erosions contained some CGRP immunoreactive fibers, perhaps as a sign of attempts of reactive repair. Our results suggest a local delivery system of potent peptide regulatory factors in bone, a system also affected by the pathophysiology of arthritis.


Digital sympathectomy for refractory Raynaud's phenomenon in an adolescent. We describe the case of an adolescent with severe Raynaud's phenomenon refractory to conventional medical management. Her course was complicated by fingertip ulceration and necrosis. The localized surgical technique of digital sympathectomy was successfully used in the management of her disease, and should be considered for intractable Raynaud's phenomenon.


Excitatory amino acid receptor ligands. Synthesis and biological activity of 3-isoxazolol amino acids structurally related to homoibotenic acid. The 3-isoxazolol amino acid (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 2) and the isomeric compound (RS)-2-amino-3-(3-hydroxy-4-methylisoxazol-5-yl)propionic acid (4-methylhomoibotenic acid, 4a) are potent agonists at the AMPA subtype of central excitatory amino acid receptors. Using 4a as a lead structure, the amino acids 4c-e, in which the 4-methyl group of 4a is replaced by substituents of different size and polarity, were synthesized. Attempts to synthesize 4-(bromomethyl)homoibotenic acid (4f), a potential receptor alkylating agent, were unsuccessful. 4-Butylhomoibotenic acid (4c) and 4-(2-hydroxyethyl)homoibotenic acid (4e) were equipotent as inhibitors of [3H]AMPA binding (IC50 = 2 microM) and showed similar excitatory activity in the rat cortical slice preparation. 4d did not show significant affinity for AMPA receptor sites, but turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist. However, like 4c,e, 4d did not significantly affect the binding of the competitive NMDA antagonist, [3H]CPP, or the noncompetitive NMDA antagonist, [3H]MK-801. None of the amino acids 4c-e showed detectable affinity for [3H]kainic acid binding sites. Like the parent compound 4a (IC50 = 0.18 microM), 4c (IC50 = 0.18 microM), 4e (IC50 = 0.14 microM), and in particular 4d (IC50 = 0.02 microM) were effective inhibitors of calcium chloride-dependent [3H]glutamic acid binding, whereas AMPA is inactive (IC50 greater than 100 microM) in this binding assay. Thus, 4d is an effective and highly selective inhibitor of calcium chloride-dependent [3H]glutamic acid binding and may be a useful tool for studies of the physiological relevance and pharmacological importance of this binding affinity.


Identification of a new DMD gene deletion by ectopic transcript analysis. The detailed genetic analysis of the Duchenne/Becker muscular dystrophy gene is hindered by the large number of exons involved and their separation by huge introns. These problems can be overcome by the analysis of mRNA rather than genomic DNA and ectopic transcripts derived from peripheral blood lymphocytes provide a convenient source of material. Using reverse transcription and nested PCR, we show here a comprehensive strategy for the rapid and complete analysis of the coding sequences from complex genes and illustrate its potential by the identification of a hitherto undescribed single exon deletion.


Shared antigenic epitopes of the major core proteins of human and simian immunodeficiency virus isolates. Antigenic epitopes on the major core (gag) protein of isolates of simian and human immunodeficiency virus (SIV and HIV) were compared using a panel of eleven mouse monoclonal antibodies (Mabs) that recognized nine distinct gag epitopes. Viral isolates used for comparison were HIV-1IIIb, HIV-2ROD, and SIV isolates from macaque (SIVmac), sooty mangabey (SIVsm-UCD), African green monkey (SIVagm), and stump-tailed macaque (SIVstm-UCD). The relatedness of the various HIV and SIV isolates, as determined by Mabs to core protein epitopes, paralleled that ascertained by genetic sequencing.


Outwardly rectifying chloride channels in lymphocytes. Outwardly rectifying Cl- channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1-6 min at +80 mV, 20 degrees C). The single-channel current at +80 mV was 4.5 +/- 0.3 pA and at -80 mV, it was 1.0 +/- 0.4 pA. After activation, the probability of being open (Po) for the lymphocyte channel was voltage independent. Activation of the Cl- channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20 degrees C exhibited Cl- channel activity. In contrast, 49% of recordings made at 30 degrees C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30 degrees C than at 20 degrees C. There was no marked change in the single-channel conductance at 30 degrees C. Open-channel conductance was blocked by 200 microM indanyloxyacetic acid (IAA) or 1 mM SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl- channels thought to be involved in fluid secretion.


Cell swelling activates K+ and Cl- channels as well as nonselective, stretch-activated cation channels in Ehrlich ascites tumor cells. Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba2+ and hence presumably to Ca2+. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca2+ permeability, as part of the volume-activated signal system. In isolated inside-out patches a Ca(2+)-dependent, inwardly rectifying K+ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mM K+ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K+ channel takes place after an increase in Ca2+ from 10(-7) to 10(-6) M which is in the physiological range. Patch-clamp studies in cell-attached mode show K+ channels with spontaneous activity and with characteristics similar to those of the K+ channel seen in excised patches. The single-channel conductance for outward current at 5 mM external K+ is estimated at about 7 pS. A K+ channel with similar properties can be activated in the cell-attached mode by addition of Ca2+ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K+ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K+ channels following addition of Ca2+ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K+ channels in the presence of 3 mM Ca2+ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca2+ and could not be activated by membrane stretch (suction). In cell-attached mode single-channel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca2+ plus ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)


Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions. The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.


Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors. The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli. The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation. The leader mRNA consists of four structural domains. The present work shows that mutations in these four domains affect expression and/or regulation in different ways. Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding. Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader. Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm. In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control. The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr). The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4. Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation. Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.


Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures. A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.


Gene structure of human CD59 and demonstration that discrete mRNAs are generated by alternative polyadenylation. We have isolated the CD59 gene from human genomic libraries. The gene is distributed over more than 27 x 10(3) base-pairs and consists of one 5'-untranslated exon and three coding exons. The gene structure is similar to that of mouse Ly-6 with the exception of the larger size of CD59 introns. Northern blot analysis using six different probes located in the 3'-region of the gene shows that more than four different CD59 mRNA molecules are generated by alternative polyadenylation. Three of these polyadenylation sites were predicted from previously published cDNA sequences. We have isolated a fourth from Jurkat poly(A)+ RNA by the procedure of rapid amplification of cDNA ends. Alternative polyadenylation may be due to the RNA secondary structure around the typical polyadenylation signal, AAUAAA.


Neuronal maps of the frontal ganglion of the cockroach, Periplaneta americana, prepared by heavy metal iontophoresis. Neurons in whole mount preparations of the frontal ganglion (FG) of the cockroach, Periplaneta americana, were mapped with the aid of cobalt chloride staining and silver intensification techniques. Eighty-six neurons were counted in the FG after staining with reduced methylene blue. The cell size ranged between 20 to 35 microns in diameter. Of the somata located in the FG, 44 were found to contribute their fibers to the nervus recurrens, 26 to the right frontal commissure, 28 to the left frontal commissure, and 6 to the nervus connectivus. In addition, a few neurons presumably from the tritocerebral region also contribute their fibers in the formation of nervus connectivus. The present study has helped delineate the neuronal connections of the FG with the brain and neuroendocrine system (corpora cardiaca and corpora allata). This information will be useful in facilitating the positioning of microelectrodes in our future electrophysiological experiments.


The loading of fura-2 into mitochondria in the intact perfused rat heart and its use to estimate matrix Ca2+ under various conditions. When rat hearts were perfused with a medium containing 10 microM fura-2/AM for 1 hr at 37 degrees C a significant amount of the derived fura-2 could be detected in subsequently isolated mitochondria. This procedure allowed the measurement of matrix free Ca2+ concentration ([Ca2+]m) of mitochondria rapidly isolated from whole hearts by a method which avoids artefactual redistribution of Ca2+. [Ca2+]m in mitochondria prepared from control hearts and incubated with respiratory substrates and EGTA was found to be 172 +/- 23 nM (mean +/- S.E.M.). When hearts were subjected to either increased mechanical work or treatment with 1 microM L-epinephrine (for 2 mins) [Ca2+]m increased to 916 +/- 138 nM and 727 +/- 65 nM respectively. The presence of ruthenium red (2.5 microM) in the perfusion medium prior to and during inotropic intervention diminished these increases in [Ca2+]m(to 316 +/- 28 nM and 218 +/- 18 nM respectively) but did not affect control values. Addition of Na+ ions to incubated mitochondria to enhance mitochondrial Ca2+ egress diminished these increases in [Ca2+]m due to pre-treatment with positive inotropes (compared to controls). These changes in [Ca2+]m were broadly parallelled by changes in the active non-phosphorylated form of pyruvate dehydrogenase (PDH) under all circumstances. These results provide further evidence that the activation of PDH by positive inotropes is accomplished by, and at least in part due to, raised mitochondrial matrix free [Ca2+] and that such increases can be maintained in isolated and suitably incubated mitochondria.


The response of rubrospinal neurons to axotomy at different stages of development in the North American opossum. Rubral axons can grow around a lesion of their pathway in the thoracic spinal cord of developing opossums and a critical period exists for that plasticity. The critical period probably begins when rubral axons first grow into the thoracic cord, and it extends until approximately postnatal day 30. We previously noted that most rubrospinal neurons die after transection of their axon during the critical period, suggesting that plasticity results primarily from growth of axons not damaged by the lesion (Xu and Martin, J. Comp. Neurol. 279, 368-381, 1989). That observation led us to study the response of rubrospinal neurons to axotomy in more detail and at additional stages of development, using a prelabeling paradigm. We first injected fast blue (FB) into the caudal thoracic or rostral lumbar spinal cord in animals ranging from estimated postnatal day 9 to 50 and, about 4 days later, lesioned the rubrospinal tract several segments rostral to the injection. Approximately 30 days later, the animals were killed so that the red nucleus could be searched for labeled neurons. During the critical period for plasticity, rubrospinal neurons showed signs of degeneration 1 week after their axon was cut. When animals were killed 2-3 weeks after lesioning, there was an obvious decrease in axotomized neurons within the red nucleus, and by 4 weeks, more than 75% of them had degenerated. The marked susceptibility of rubrospinal neurons to axotomy during the critical period for plasticity is consistent with the hypothesis that developmental plasticity of the rubrospinal tract results primarily from growth of axons that were not damaged by the lesion. Our results also suggest that survival of axotomized rubrospinal neurons increases with age.


Toxicity to rats of methanol-fueled engine exhaust inhaled continuously for 28 days. Fischer 344 rats were exposed to three concentrations of exhaust generated by an M85 methanol-fueled engine (methanol with 15% gasoline) without catalyst for 8 h/d, 7 d/wk for 7, 14, 21, or 28 d. Concentration- and time-dependent yellowing of the fur was prominent in all treated groups. Concentration-dependent increases in the erythrocyte count, hematocrit, hemoglobin concentration, formaldehyde in plasma, and carboxyhemoglobin in the erythrocytes, and decrease in serum alkaline phosphatase activity were seen after all exposure periods. Histopathologically, lesions were found in the nasal cavity and lungs after 7 d of exposure. Squamous metaplasia of the respiratory epithelium of level 1 (level of the posterior edge of the upper incisor teeth) lining of the nasoturbinate and/or maxilloturbinate and infiltration of neutrophils into the submucosa, and decreases of Clara cells in the terminal bronchiolus and of cilia in the bronchiolar epithelium, were observed in the high-concentration group (carbon monoxide, 94 ppm; formaldehyde, 6.9 ppm; methanol, 17.9 ppm; nitrogen oxides, 52.7 ppm; nitrogen dioxide, 10.6 ppm). The histopathological extents of several lesions increased slightly with the exposure time. Slight squamous metaplasia and hyperplasia of the respiratory epithelium at level 1 were also observed in the medium-concentration group (one in three of the high-concentration group). No histopathological changes were found in the olfactory epithelium of the nasal cavity. In the low-concentration group (one in nine of the high-concentration group), no marked histopathological changes in these organs were observed. These results may suggest that the lesions observed in the nasal cavity of rats exposed to methanol-fueled engine exhaust were mainly caused by formaldehyde, although other components in the exhaust also may have affected nasal cavity and/or lungs to less extent.


Axons modulate the expression of proteolipid protein in the CNS. We examined the expression of mRNA encoding proteolipid protein (PLP), the major myelin protein in the CNS, in developing rat cerebrum, and in normal and degenerating optic nerves. PLP transcripts were initiated at two clusters of start sites that were separated by about 30 base pairs. During the peak of PLP mRNA expression in developing cerebrum, a higher proportion of PLP transcripts were initiated from the distal start site, furthest from the open reading frame, than in mature cerebrum. We enucleated one eye of immature rats to cause Wallerian degeneration in the optic nerve. In these degenerating optic nerves, the steady state levels of PLP mRNA fell markedly, and the proportion of distally initiated PLP transcripts declined to the same proportion found in normal adult nerves. Changes in myelin gene expression were not limited to PLP mRNA, as the steady-state levels of myelin basic protein (MBP) mRNA paralleled those of PLP mRNA in the developing cerebrum and in degenerating optic nerves. Thus, oligodendrocytes require axons to maintain their normal levels of PLP and MBP transcripts and the high proportion of distally initiated PLP transcripts that characterize early myelination.


Interaction between oligodendroglia and immune cells: mitogenic effect of an oligodendrocyte precursor cell line on syngeneic T lymphocytes. We analyzed cellular interactions between T lymphocytes and a recently established immortal glial line, L3 that retains several properties of immature oligodendrocytes (Aloisi et al., J Neurosci Res 27:16-24, 1990). L3 oligodendrocytes (L3-OL) cannot be induced to express class II antigens, nor do they specifically present antigen to syngeneic specific T lymphocyte. However, L3-OL strongly enhance the proliferation of freshly activated, interleukin-2(IL-2)-dependent T-line lymphocytes and concanavalin A (ConA)-activated lymphoblasts, irrespective of their antigen specificity or surface phenotype (CD4+ or CD8+). Resting and some activated T cells were susceptible to the mitogenic effect of L3-OL only in the presence of exogenous IL-2, not of other cytokines. The mitogenic effect of L3-OL did not depend on cell viability. It was observed in paraformaldehyde-fixed L3-OL cells and in membrane preparations, but not in culture supernatant. Neither intact L3-OL cells nor membrane preparations had direct IL-2 activity. The conclusion that the mitogenic effect of L3-OL cells is exerted by membrane structures acting as a costimulatory factor(s) of IL-2 is supported by the finding that it is largely blocked by a monoclonal anti-IL-2 receptor antibody. The effect is distinct from membrane-bound IL-1, membrane-bound tumor necrosis factor-alpha (TNF-alpha), IL-3, or IL-6 and cannot be reconstituted by these cytokines.


AP-1 complex and c-fos transcription are involved in TPA provoked and trans-synaptic inductions of the tyrosine hydroxylase gene: insights into long-term regulatory mechanisms. We have previously shown that the phorbol ester, TPA, which activates protein kinase C, causes, in PC12 cells, a transcriptional activation of tyrosine hydroxylase (TH), the key enzyme in catecholamine synthesis. The study has now been extended to examine the processes that underlie this transcriptional stimulation and, in addition, to seek whether similar mechanisms are involved in long-term trans-synaptic induction of the TH gene in adrenal medullae of rats that have been given a single injection of reserpine. In both systems, it was found that the induction of c-fos gene transcription was associated with that of the TH gene but with different kinetics. The promoter of the TH gene contains (at position -207/-200) a sequence (TGATTCA) which differs from the consensus TRE or AP-1 site (TGACTCA) by one nucleotide. Experiments were carried out to investigate whether the AP-1 protein complex which is known to contain Fos and Jun binds to the putative TRE region of the TH promoter. In the gel shift assays, the nuclear protein extracts derived from TPA-treated PC12 cells and from AM of reserpine injected rats displayed a higher magnitude of binding to a 25-mer TRE-TH oligonucleotide as compared to controls. The results showed that the behaviour of TRE-TH was atypical in that two retarded complexes (A and B) were observed, which were displaced by specific competitors. Trans-activation experiments with plasmids TRE-TH/TK/CAT and -754/-19 TH/pUC18-CAT in PC12 cells showed an increase in CAT activity in response to TPA that correlates with the previously observed increase in TH transcriptional activity by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)


The nonmyristylated Pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55gag and is incorporated into viruslike particles. The expression of the pol gene of human immunodeficiency virus type 1 occurs via a ribosomal frameshift between the gag and pol genes. The resulting protein, a Gag-Pol polyprotein, is produced at a level 5 to 10% of that of the Gag protein. The Gag-Pol polyprotein is incorporated into virions and provides viral protease, reverse transcriptase, and integrase, which are essential for infectivity. It is generally believed that the Gag-Pol polyprotein is incorporated into virions via interaction with the Gag protein, although the details of the mechanism are unknown. To further study this problem, we have constructed a human immunodeficiency virus type 1 proviral genome which overexpresses the Gag-Pol polyprotein (Pr160gag-pol). Transfection of this proviral genome (pGPpr-) into COS-1 cells resulted in the expression of full-length Pr160gag-pol polyprotein. Although the majority of the Pr160gag-pol was confined to the cells, low levels of reverse transcriptase activity were detectable in the cell supernatants. The cotransfection of pGPpr- with a second plasmid which expresses only the Pr55gag precursor (pGAG) resulted in a significantly higher level of Pr160gag-pol in the medium of transfected cells. Sedimentation analysis using sucrose density gradients demonstrated that most Pr160gag-pol was found in fractions corresponding to the density of virion particles, indicating that the Pr160gag-pol polyprotein was released in association with a Pr55gag viruslike particle. To further characterize the requirements for the release, a mutation was constructed to express an unmyristylated Pr160gag-pol polyprotein. Coexpression with Pr55gag demonstrated that the unmyristylated Pr160gag-pol was also incorporated into virion particles. Subcellular fractionation experiments revealed that the distributions of the Pr160gag-polmyr- and Pr160gag-pol in the membrane and cytosol were similar under low- or high-ionic-strength conditions. Taken together, these results suggest that myristylation of the Pr160gag-pol polyprotein is not required for the interaction with the Pr55gag necessary for packaging into a viruslike particle.


Mutagenic analysis of the coronavirus intergenic consensus sequence. Previously, a system in which an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells was established. In the present study, a DI cDNA containing one UCUAAAC consensus sequence in the middle of the 0.3-kb-long intergenic region located between genes 6 and 7 was constructed. From this DI cDNA clone, 21 mutant DI RNAs were constructed so that each of the seven consensus sequence nucleotides was changed individually to the three alternative bases. These mutants were used to define how changes in the integrity of MHV transcription consensus sequence UCUAAAC affected mRNA transcription. Except for two mutants with the sequences UGUAAAC and UCGAAAC, all of the mutants supported efficient subgenomic DI RNA transcription. This indicated that MHV transcription regulation was sufficiently flexible to recognize altered consensus sequences. Next, these and other mutants were used to examine the leader-body fusion site on the subgenomic DI RNAs. Sequence analysis demonstrated that all subgenomic DI RNAs analyzed contained two pentanucleotide sequences; the first sequence seemed to be contributed by the leader, and the leader-body fusion most likely took place at either the first or the second nucleotide of the second sequence. This observation was not consistent with the proposed coronavirus transcription model (S. C. Baker and M. M. C. Lai, EMBO J. 9:4173-4179, 1990) which states that nucleotide mismatch can be corrected by RNA polymerase proofreading activity.


Requirements for strand transfer between internal regions of heteropolymer templates by human immunodeficiency virus reverse transcriptase. We have examined the ability of the reverse transcriptase (RT) from human immunodeficiency virus (HIV) to carry out strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions of natural-sequence RNA. A 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide was used to initiate synthesis. DNA oligonucleotides with homology to internal regions of the donor were used as acceptors. In this system, HIV RT produced strand transfer products. An HIV RT having RNase H depleted to 3% of normal (HIV RTRD) catalyzed the transfer reaction inefficiently. An RNase H-minus deletion mutant of murine leukemia virus RT was unable to catalyze strand transfer. HIV RTRD, however, efficiently catalyzed transfer when Escherichia coli RNase H was included in the reactions, while the mutant murine leukemia virus RT was not efficiently complemented by the E. coli enzyme. Evidently, RNase H activity enhances, or is required for, internal strand transfer. Two acceptors homologous to 27-nucleotide regions of the donor, one offset from the other by 6 nucleotides, were tested. The offset eliminated a sequence homologous to a prevalent DNA synthesis pause site in the donor. Strand transfer to this acceptor was about 25% less efficient, suggesting that RT pausing can enhance strand transfer. When the deoxynucleoside triphosphates in the reactions were reduced from 50 to 0.2 microM, increasing RT pausing, the efficiency of strand transfer also increased. A model for RT-catalyzed strand transfer consistent with our results is presented.


Persistence of viral RNA in mouse brains after recovery from acute alphavirus encephalitis. Little is known about the relationship between recovery from acute viral encephalitis and the clearance of viral genetic material from the central nervous system. In a mouse model of Sindbis virus encephalitis, we have previously shown that clearance of infectious virus is mediated by antibody-induced restriction of viral gene expression rather than by cytotoxic destruction of virally infected cells. To explore whether Sindbis virus genomes persist in mouse brain after the clearance of infectious virus, we used reverse transcriptase-polymerase chain reaction amplification methods to detect Sindbis virus RNA in brain samples from immunocompetent BALB/c and antibody-treated immunodeficient scid/CB17 mice. RNA sequences from both the nonstructural region (NSP1 gene) and structural regions (E2 gene) of Sindbis virus were detected in the brains of all BALB/c and antibody-treated scid mice examined at 1, 2, and 3 months after infection. Additional BALB/c mouse brains were also positive at 8, 12, and 17 months after infection. To determine whether persistent RNA was capable of resuming unrestricted replication in the absence of the continuous presence of antiviral antibodies, viral titers were measured in the brains of scid mice at 1, 2, 3, and 6 months after antibody treatment. Viral reactivation was seen in scid mice treated with hyperimmune serum or a low dose of monoclonal antibody to the E2 envelope glycoprotein, but not in mice treated with a high dose of monoclonal antibody to E2. Replication of infectious virus isolated from scid mouse brain could be restricted by repeat treatment with immune serum, indicating that viral reactivation is not due to antibody-escape mutations. These results demonstrate that Sindbis virus can persist long term in a nonproductive form in mouse brain and suggest that the humoral immune response plays an important role in preventing viral reactivation.


Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope. Studies with molecular and immunological techniques identified and mapped the transcript encoding glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1 nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma) transcript. The 3.8-kb gD mRNA initiates within the US segment 91 and 34 nucleotides downstream of the CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum prior to infection results in reduced numbers of plaques and reduced levels of virus-encoded thymidine kinase. Complement is not required for neutralization mediated by the anti-19-mer serum.


Epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus. The epitope specificity of the protective immune response against swine transmissible gastroenteritis (TGE) has been investigated by using circulating and secretory antibodies. This study was carried out with sows vaccinated with TGEV or the antigenically related porcine respiratory coronavirus (PRCV). TGEV vaccination of sows resulted in greater lactogenic protection of suckling piglets against TGEV challenge and a higher secretory immune response than PRCV vaccination did. These differences in the immune response were conditioned by the route of antigen presentation as a result of the different tropism of each virus. Epitopes on S protein, and in particular those contained in its antigenic site. A, were more immunogenic than epitopes on N and M proteins in both groups of vaccinated sows, as determined by a competitive radioimmunoassay. Minor differences in antibody response against the previously defined antigenic subsites Aa, Ab, and Ac were also detected, with subsite Ab being the most antigenic in both TGEV- and PRCV-immune sows. These findings suggest that antigenic site A on S protein, involved in virus neutralization, is the immunodominant site in pregnant sows that confer lactogenic protection. They also validate, in experiments with secretory antibodies, the antigenic maps made with murine monoclonal antibodies. Therefore, this antigenic site should be considered for vaccine or diagnostic development.


A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes. Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.


Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus. Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26- to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and all but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin G1 subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the N- or M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE.


Design of high-affinity major histocompatibility complex-specific antagonist peptides that inhibit cytotoxic T-lymphocyte activity: implications for control of viral disease. Cytotoxic T lymphocytes (CTLs) recognize viral antigens presented by infected cells in the context of their major histocompatibility complex glycoproteins. The irreversible killing of virus-infected cells by virus-specific CTLs can be the cause of serious disease, particularly in the central nervous, hepatic, and cardiovascular systems. Design of molecules controlling (blocking) interaction between CTLs and infected cells, and their further use to inhibit (or antagonize) T-lymphocyte activity, is an important pharmacologic goal. In this report, we describe the design of a new family of peptides which selectively inhibit activity of lymphocytic choriomeningitis virus-specific CD8+ T lymphocytes, which recognize endogenously processed viral epitopes presented by major histocompatibility complex class I molecules.


Identification of T-cell epitopes on E2 protein of rubella virus, as recognized by human T-cell lines and clones. T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell cloned isolated from one T-cell line responded strongly in proliferation assays with peptide E2-4 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HLA DR7.


In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.


Use of the ASI titanium stent in the management of bladder outflow obstruction due to benign prostatic hyperplasia. An expandable titanium intraprostatic stent was inserted into 30 patients with infravesical obstruction due to benign prostatic hyperplasia (BPH). All of the men were considered unsuitable for transurethral resection of the prostate as a result of comorbid conditions. In 25 patients effective micturition was reestablished with this technique. In 21 of these men, who have been followed for longer than 1 year, the mean maximum flow rate at 1 year was 10.8 ml. per second and the mean residual urine was 56 ml. Although urinary tract infections occurred subsequent to stent insertion in 10 individuals, these resolved after appropriate antibiotic treatment and no stents have had to be removed for this reason. Followup cystoscopy or examination by electron microscopy of those stents that have been removed has shown partial epithelialization of the stent surface in a proportion of patients, and a minor degree of incrustation occurred in 1 case. We conclude that an expandable intraprostatic titanium stent is an acceptable alternative to transurethral resection of the prostate or long-term catheterization in this particular group of high risk patients.


Multicenter, randomized, double-blind, placebo controlled study to investigate the effect of finasteride (MK-906) on stage D prostate cancer. A total of 28 untreated patients with asymptomatic, stage D prostate cancer was randomized in a double-blinded fashion to receive finasteride (10 mg. per day), a 5 alpha-reductase inhibitor or placebo. Patients were evaluated at 3-week intervals by rectal examination, and serum prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) levels, and at 6-week intervals by bone scan and transrectal ultrasound determinations of prostatic volume. Patients stopped the medication at week 6 at the discretion of the investigator when PSA levels increased from baseline. After 12 weeks all patients were reevaluated. Of the patients 13 received finasteride and 15 received placebo. The 2 groups did not differ statistically with respect to patient age, initial PSA and PAP level, or the extent of metastases on initial bone scan. A statistically significant decrease in the median percentage change from baseline in PSA at weeks 3 and 6 occurred in the finasteride group compared to the placebo group (-22.9% versus -2.9% and -15.1% versus +11.7%, respectively, p less than 0.05). Finasteride had no effect upon PAP, serum testosterone, prostatic volume or appearance of bone scans. A decrease in serum PSA in the finasteride treatment group suggests that finasteride exerts a minor effect in patients with prostate cancer. This effect does not approach that seen with medical or surgical castration yet because of the potency preserving feature and the lack of toxicity finasteride may warrant further study in the treatment of prostate cancer.


Impact of radical prostatectomy on the characteristics of bladder and urethra. A prospective study was done to evaluate the long-term effects of radical prostatectomy on the function of the bladder in filling and voiding. Preoperative urodynamic studies were done on 29 patients with a mean age of 62.9 +/- 5.2 years. The preoperative results show that 16 of the 29 patients demonstrated detrusor instability with maximum contractile pressures of 59 +/- 28 cm. water. Followup urodynamic assessment was done in 13 of these patients 22.9 +/- 1.1 months after surgery. Postoperatively, the maximum detrusor instability pressure did not decrease significantly (49 +/- 17 cm. water). Comparison of the operative and postoperative urodynamic characteristics of bladder filling shows that radical prostatectomy produced no significant change in the filling characteristics of the bladder in terms of bladder capacity, or volume at which sensations of fullness or urgency are reported. Voiding pressure-flow studies show a significant increase in maximum flow rate (8 +/- 1 to 13 +/- 2 ml., per second, p = 0.007), and significant decreases in maximum detrusor pressure (61 +/- 5.4 to 39 +/- 4 cm. water, p = 0.002), urethral opening pressure (45 +/- 7 to 25 +/- 4 cm. water, p = 0.004) and residual volume (150 +/- 37 to 62 +/- 43 ml., p = 0.019). Urethral profile measurements show that there was no significant change in either the maximum urethral closure pressure (94 +/- 9 to 83 +/- 9 cm. water) or external sphincter length (3.6 +/- 0.8 to 3.2 +/- 0.8 cm.). Preoperatively, the bladder neck pressures were 25 +/- 4.4 cm. water and were abolished after prostatectomy, indicating that the decrease in obstructive characteristics is due to removal of the prostate.


Lewis blood group antigen expression by cultured normal ureteral epithelial cells. The importance of Lewis blood group antigens in recurrent urinary tract infections has led to a more detailed study of the expression of these antigens in an in vitro culture system. Expression of A, B, H, and Lewis blood group antigens by primary cultures of ureter epithelial cells was analyzed by flow cytometry. Cells maintained blood group determinants for up to 7 weeks in culture over three passages. In several specimens, the cells that exhibited expression of Lewis A (Le(a) and Lewis B (Le(b)) antigens increased dramatically over 2-3 passages in culture. Lewis Y (Le(y)) antigen expression was positive on all primary cultures tested. The effect of fetal bovine serum (FBS) on blood group antigen expression was evaluated. Increased concentrations of FBS in the growth media increased the expression of Le(a) and Le(b) antigens in certain specimens, but did not affect expression of the other blood group antigens. The effect of FBS on blood group antigen expression of cultured cells depended on the Lewis blood type of the individual donating the specimen. To evaluate the heterogeneity often seen in Le(a) and Le(b) antigen expression, dual staining of cells for Le(a) and Le(b) antigens was performed. The results of these studies show for the first time that Lewis blood group antigens are expressed by cultured normal ureteral epithelial cells and that the pattern of expression of Le(a) and Le(b) changes over time. Furthermore, an individual cell may simultaneously express more than one Lewis antigen on its surface.


Beta-3 adrenergic agonist, BRL-26830A, and alpha/beta blocker, arotinolol, markedly increase regional blood flow in the brown adipose tissue in anesthetized rats. Regional vascular effects of some adrenergic agents, focussing on brown adipose tissue (BAT), were investigated using tracer microspheres with a reference sample method in the anesthetized rat. Intravenous injections of 0.5 mg/kg BRL-26830A, a beta 3-adrenergic agonist, increased heart rate, but changes in blood pressure and cardiac output were not significant. The drug decreased blood flow in the brain, the spleen and the kidneys, but markedly increased it in BAT. At 2 mg/kg, arotinolol, an alpha/beta-adrenergic blocker, decreased blood pressure by 20 mmHg and increased cardiac output by 95 ml/min/kg. It slightly but significantly decreased blood flow in the liver and the spleen, but markedly increased the flow in BAT. Acebutolol, a beta 1-adrenergic blocker, decreased blood flow in the liver, the spleen, the pancreas, the kidneys, the adrenals, the skeletal muscle and the skin. Bunazosin, an alpha 1-adrenergic blocker, decreased it in all organs and tissue expect the brain and BAT. The pattern of redistribution of blood flow by arotinolol was very similar to that caused by BRL-26830A. Acebutolol and bunazosin rather decreased the blood flow in the BAT. These results indicate that stimulation of beta 3-adrenergic receptors, in BAT results in vasodilation, and that arotinolol may bind to those beta 3-adrenergic receptors.


A case of vasovagal syncope with convulsions--the effects of midodrine hydrochloride. A 42-year-old female had suffered from repeated syncope. She had vasovagal syncope with convulsions from vasodilatation and cardiac standstill which lasted for 9.8 sec. The 60 degrees head-up tilt test, nitroglycerin injection and isoproterenol infusion provoked vasovagal reaction. Although a beta blocker was not effective in preventing tilt-induced hypotension and bradycardia, midodrine hydrochloride (alpha-1 stimulant) or atropine prevented it. In this patient, insufficient constriction of capacitance vessels might have played an important role in activation of an inhibitory reflex from cardiopulmonary mechanoreceptors which caused hypotension and bradycardia.


[CD7 (+) stem cell leukemia presenting different phenotypes in lymph node and bone marrow]. A 27-year-old male with systemic lymphadenopathy was diagnosed as lymphoblastic-type lymphoma by inguinal lymph node biopsy in September, 1990. Bone marrow at the initial diagnosis contained 55.4% lymphoblasts with a phenotype of peroxidase (-), CD7 (+), CD4 (-), CD8 (-). Lymphadenopathy and lymphoblasts in bone marrow disappeared after MACOP-B therapy. In December, 1990, however, the patient again noticed swelling of cervical lymph nodes. At this time, the bone marrow contained 36.4% myeloblasts with a peroxidase (+), CD7 (+), CD13 (+), CD33 (+) phenotype. Cytogenetic and genetic study revealed that the lymphoblasts at the initial diagnosis and the myeloblasts at relapse shared an common abnormal karyotype, 11p-, and the same rearranged band of T-cell receptor delta, gamma, beta genes, suggesting that these two blasts originated from the same clone. The blasts obtained from the cervical lymph node at relapse were still negative for peroxidase, in contrast to the blasts from bone marrow. These findings suggest that this leukemia originated from a stem cell and differentiated along multilineage pathways.


Immunohistochemical classification of astrocytes in infants by glial fibrillary acidic protein staining and application to forensic practice. This study was designed to assess whether development of the astrocytes in central nervous system (CNS) is delayed in victims of the sudden infant death syndrome (SIDS), and to try the classification of astrocytes by a morphological method. The glial fibrillary acidic protein (GFAP) is the major component of astrocytic fiber and is characteristic of astrocytes in the human CNS. An immunohistochemical study identificating this protein was carried out on infant brains to evaluate astrocytes. Morphologically 39 brains of infants aging under 12 months old, that including 10 asphyxia, 13 respiratory infections and 16 SIDS cases, were stained for GFAP by the peroxidase-anti-peroxidase (PAP) method. According to their staining pattern astrocytes were classified into 4 cell types: Type I cells were seen during the non-proliferating period, Type II cells during the low-proliferating period, Type III cells during the moderate-proliferating period and Type IV cells during the high-proliferating period. On the basis of this classification of astrocytes, it was observed that the first and most reactive pattern of physiological proliferation was in infant brains four months old after birth in the asphyxia cases. The staining grade of astrocytes was significantly less in white matter in the SIDS victims compared with age-matched of the respiratory infections and asphyxia. Therefore, we suggest that development of brain in the SIDS cases is delayed to controls and that the SIDS is associated with a developmental CNS disorder. Among 3 group cases the retarded staining pattern of the astrocytes in SIDS is interesting and might give a helpful approach to diagnosis of the cause of death for SIDS.


Effects of adrenergic agonists on an experimental urinary incontinence model in anesthetized rabbits. We have developed an experimental urinary incontinence model in anesthetized female rabbits, in order to study the effects of alpha-adrenergic receptor agonists on it in vivo. Micturition was induced artificially by electrical stimulation of the abdomen of rabbits receiving a continuous infusion of glucose-free. Tyrode's solution into the urinary bladder. Alpha-1 adrenergic agonists, phenylephrine (1 mg/kg, i.v.) and the newly synthesized agent ST-1059 (1 mg/kg, i.v.) and its prodrug midodrine (10 mg/kg), which was intraduodenally administered, elevated the bladder pressure and arrested micturition induced by electrical stimulation. Prazosin (0.1 mg/kg, i.v.) inhibited these effects of phenylephrine. The effect of an alpha-2 agonist, clonidine (1 mg/kg, i.v.), on micturition induced by electrical stimulation was not clearly defined. This study demonstrates that alpha-1 adrenergic agonists can arrest artificially-induced micturition via urethral contraction. This method may be useful for evaluating the effect of a drug on urethral leakage in vivo.


[Nosocomial respiratory infection caused by Pseudomonas cepacia in immunocompromised hosts]. Pseudomonas cepacia is a gram negative rod, having no fermentative activity on glucose. This organism was detected in the sputum, throat swab, or throat washing of 22 inpatients treated between January, 1990, and December, 1990, at the First Department of Internal Medicine, Kagawa Medical School. The primary diseases for which these 22 patients were hospitalized were leukemia in 12, malignant lymphoma in 5, lung cancer in 2, myelodysplastic syndrome in 1, and embryonal cell carcinoma in 1. Twelve of the 22 patients had episodes of pneumonia which complied clinically with the diagnostic criteria provided to facilitate the National Nosocomial Infection Study. The complication of pneumonia occurred in 7 patients with leukemia, 2 with malignant lymphoma, 2 with lung cancer, and 1 with myelodysplastic syndrome. In 10 of these 12 patients, the organism was detected before the onset of pneumonia. All 22 patients in whom the organism was demonstrated had received antibiotics. The antibiotics which was most frequently used to treat these patients 1 month before detection of Pseudomonas cepacia were amikacin and ceftizoxime, which were used in 13 patients. Of the antibiotics in which the susceptibility to Pseudomonas cepacia was, evaluated, minocycline was effective in 100% (21/21), ceftazidime in 50% (11/22), and ofloxacin in 27.3% (6/22). Physicians should be especially aware of the possibility of colonization and nosocomial respiratory infection by Pseudomonas cepacia in patients with severe underlying diseases.