Effects of a nitric oxide synthase inhibitor on non-cholinergic junction potentials in the circular muscle of the guinea pig ileum. Intracellular microelectrodes were used to record junction potentials from the circular muscle cells of the guinea pig ileum in vitro at 37 degrees C in a modified Krebs solution containing nifedipine (1-2 microM) and hyoscine (1 microM). Transmural nerve stimulation, using volleys of three pulses at 50 Hz, produced a complex response consisting of an inhibitory junction potential (IJP) followed by a prolonged depolarization. Following the addition of the nitric oxide synthase inhibitor NG-nitro-L-arginine (NOLA, 100 microM) the amplitude of the IJP (recorded 10 mm aboral to the stimulating electrodes) was increased by approx. 10% (n = 4). The further addition of apamin (250 nM) abolished the IJP revealing a non-cholinergic excitatory junction potential (EJP). In other experiments (n = 8), preparations were treated with apamin then subjected to substance P desensitization (500 nM, > 20 min). Transmural nerve stimulation now produced a triphasic response (recorded 1 mm aboral to the stimulating electrodes) consisting of: (a) an initial hyperpolarization (approx. 5 mV) lasting about 1 s; followed by (b) a depolarization reaching a peak (approx. 7 mV less negative than the RMP) approx. 2 s after nerve stimulation; and finally (c) a small (approx. 3 mV) hyperpolarization. The addition of NOLA reduced all three phases by 80-90% (n = 8). The subsequent addition of L-arginine (5 mM) partially reversed these effects (n = 3). Conditioning hyperpolarization up to 20 mV increased the amplitude of the NOLA-sensitive IJP and EJP. Further conditioning hyperpolarization reduced the amplitude of the IJP and enhanced the amplitude of the EJP. Large conditioning hyperpolarizations (> 60 mV) reduced the amplitude of both the IJP and EJP. An estimation of the membrane conductance changes occurring during the initial hyperpolarization and depolarization suggest that it was either unchanged or increased. During large conditioning hyperpolarizations in the absence of nerve stimulation, the membrane potential was unstable and began to show spontaneous oscillations (up to 30 mV, every 4-5 s) resembling slow waves. These experiments indicate that NO, or a related compound, appears to mediate the nerve induced apamin-resistant IJP and substance P- and hyoscine-resistant EJP in the circular muscle of the guinea pig ileum.


Pancreatic enzymes in chronic renal failure and transplant patients. The aim of the present study was to determine the frequency and degree of elevated serum levels of Total Amylase (TA), Pancreatic Amylase (PA), and Lipase (L) activity in patients with chronic renal failure (CRF) on conservative therapy; CRF on periodical hemodialysis (HD); in renal transplant (RT) and in a control Group (C). Mean values were significantly higher in all groups than Group C for TA (p < 0.005), PA (p < 0.0001) and L (p < 0.0001). A statistically significant correlation was found between TA and L vs creatininemia values in CRF patients, but only up to a certain level (creatininemia < 6 mg %) (p < 0.03 and p < 0.05), above which there was no correlation. The enzyme most frequently over the maximum normal limit was PA, both in the total CRF group (51%), in the hemodialysis patients (65%), and in the RT patients (55%); but only a few patients had values two times higher than the normal limits: 15% in the total CRF, 14% is the hemodialysis, and 10% in the RT groups, respectively. These results suggest that the increase in serum pancreatic enzyme during chronic renal pathology is slight but frequently occurs. It is possible that in these patients together with the renal excretion impairment there could also be some subclinical pancreatic damage; its genesis could also depend on the pharmacological treatment used (diuretics, immunosuppressive drugs) commonly adopted in these pathologies.


Stimulation of hepatic protein synthesis in response to Mikania cordata root extract in carbon tetrachloride-induced hepatotoxicity in mice. The effect of Mikania Cordata root extract was evaluated on the rate of hepatic protein synthesis in vivo in CCl4-induced liver damage. Pretreatment with the root extract (100 mg/kg, once daily for successive 5 days) showed a marked enhancement in the levels of hepatic DNA, RNA and protein content that were adversely affected with CCl4 treatment in the experimental mice. Increase in the total protein mass, fractional rate of protein synthesis (% of protein synthesized/day), total rate of protein synthesis (fractional rate x protein mass), ribosomal capacity (RNA/protein), ribosomal efficiency (rate/ribosome) and high turnover rate of protein (protein/DNA) in response to the pretreatment of the root extract in hepatic tissue indicated the tissue repair leading to a functional improvement of the hepatocytes that were disorganised with CCl4 intoxication.


Effect of fungicides on the production of amylase by Rhizopus oryzae. Seven fungicides (Brassicol), captan, Dithane M-45, Fytolan, Parasan, Sulfex and Thiram) were tested for their effect on the production of amylase enzyme by Rhizopus oryzae. The activity of amylase was determined by cup-plate method. All the fungicides were found inhibitory for the synthesis of amylase. Out of these, Parasan was the most effective causing hundred percent inhibition at 0.025% concentration. No amylase production was recorded when Thiram, Fytolan, Brassicol, Captan and Dithane M-45 were used at 0.5, 1.5, 2.0, 2.0, and 2.0% concentration respectively. Sulfex was found to be less effective, as hundred percent inhibition of amylase production has not been recorded even at 3.0% concentration.


Evaluation of surgical risk in palliation and resection of pancreatic cancer. Perspective study and tables to calculate the risk. High morbidity and mortality rates are reported for bypass and resective surgery of pancreatic cancer. In a retrospective study we correctly predicted the postoperative course in 88% of the patients who underwent bypass surgery and 83% of those who had a resection for pancreatic cancer. Before starting with clinical application of this scoring system, we undertook a prospective study to confirm its predictive value. Sixty-seven consecutive patients with pancreatic cancer were included: 42 patients underwent bypass surgery and 25 pancreatic resections. The operative mortality was 14% for palliative surgery and 0% for resective surgery. Surgical team and nurses were totally unaware of the predicted risk. The preoperative forecast proved to be correct in 81% of bypass surgery and in 88% of resective surgery, although surgical mortality had decreased from 21 to 14% for bypass surgery and from 17 to 0% for resective surgery. Tables are included to calculate the surgical risk for each of 162 combinations of the risk factors considered in the predictive model (81 for bypass surgery and 81 for resective surgery). Calculation of surgical risk is important when evaluating different treatments for pancreatic cancer are available.


Histological pancreatitis in end-stage renal disease. To clarify a possible cause of hyperamylasemia in end-stage renal disease (ESRD), histological studies were performed on the pancreatic glands of twenty-seven autopsied patients with ESRD who had received long-term hemodialysis. The findings were compared with those in a similar number of age-matched control subjects. Histological evidence of pancreatitis was found in 51.9% of the ESRD patients as compared with 14.8% in the controls (p < 0.005). The pancreatitis was chronic in nature in 85.7% of the ESRD patients showing changes of pancreatitis. Secretin administration to an additional group of twelve patients with ESRD induced an elevation in the activities of both total and P-type serum amylase in only one patient. These findings suggest that although histological pancreatic alterations are common in patients with ESRD, they are probably not responsible for the P-type hyperamylasemia frequently found in such patients.


Genetic control of bone remodeling.

Genetic control of bone remodeling. Biological research related to orthodontics naturally leads to two general areas. One is the investigation of craniofacial growth and development in normal and abnormal circumstances, attempting to understand the etiological basis for the development of malocclusion or dentofacial deformity. The other concerns developing an understanding of the basic mechanisms involved in tooth movement during orthodontic therapy. This paper reviews ongoing research in both areas.


Targeted drug delivery.

Targeted drug delivery. Achieving adequate dosage of a drug to be effective, without crossing the line into toxicity, can be a problem. This paper reviews ongoing research into methods of delivering drugs only to diseased tissue or cells, minimally affecting surrounding tissue. In particular, the author discusses encapsulating drugs in liposomes (artificial phospholipid microcapsules) which will only bind to certain cells.


Growth factors in bones and teeth.

Growth factors in bones and teeth. Human bone contains an abundance of polypeptide growth factors. These growth factors stimulate the proliferation and activity of bone cells and can stimulate bone formation. Data from this laboratory and others suggest that bone growth factors may act to couple bone formation to resorption to maintain bone mass during remodeling. Research is underway to study these growth factors in bones and teeth, and their possible roles in both the pathogenesis and the treatments of osteoporosis and dental diseases.


Shape changes due to functional appliances. Functional appliances are now routinely used in orthodontic treatment to correct anteroposterior skeletal discrepancies, based on the assumption that existing functional patterns can be modified to yield a new morphological pattern. However, the mechanisms by which this correction occurs remain in dispute as current measurement procedures, primarily mathematical, do not adequately describe shape changes. This paper introduces a new measurement method, Fourier descriptors, to clinical dentistry.


The role of integrin adhesion receptors in gingival wound healing. Integrins, a family of cell adhesion receptors, play an important part in wound healing. These complex macromolecules are defined, and their roles explored.


Esotropia following posterior superior alveolar nerve block. Adequate use of local anesthetics is an important phase of modern dentistry. Regardless of the care used in administration of local anesthetics, unusual reactions can occur. A case is presented in which posterior superior alveolar administration of two percent Lidocaine 1/100,000 epinephrine resulted in medial rotation of the orbit (esotropia).


The effects of adenine and dimethyl sulfoxide on the mouse pancreas. The authors have studied the effects of dimethyl sulfoxide (DMSO) on the plasma alpha-amylase activity in mice that sustained a pancreatic injury induced by an oral administration of adenine. In mice given a 5% solution of DMSO as drinking water for 3 d prior to the administration of adenine (175 mg/kg), and also drank this DMSO solution until the end of the experiment, hyperemia of the pancreas was observed and the level of plasma alpha-amylase activity became significantly higher than the level seen in the control mice. A pathological examination also revealed vacuolation and zymogenic degranulation. Further, the plasma alpha-amylase activity level increased only in mice given this 5% DMSO solution, and no increase was noted in mice given a 3% or a 1% DMSO solution for drinking water. Further, the pancreatic lipid peroxide level of mice given this 5% DMSO solution was significantly higher than the level seen in the control group. Based on the above results and associated data, it is thought that an oral administration of adenine can induce a pancreatic injury in the mouse, and that this injury is sustained with the assistance of DMSO.


Mexiletine and disopyramide suppress ventricular premature contractions (VPC) irrespective of the relationship between the VPC and the underlying heart rate. The effects of mexiletine (300 mg/day, 24 patients) and disopyramide (300 mg/day, 20 patients) on ventricular premature contractions (VPCs) were studied using a 24-hour ambulatory electrocardiogram. The VPC frequency was evaluated as a function of the underlying heart rate (HR). The VPC-HR correlation was classified into 2 major types, depending on whether the frequency of the VPC increased with the increased HR (positive type) or not (nonpositive type). The effects of the drugs were assessed based on the VPC-HR correlation and on the percent reduction of the VPC frequency. Mexiletine and disopyramide significantly decreased the frequency of the VPCs of both the positive and nonpositive types. Each drug was assumed to be effective when the percent reduction of the VPC frequency exceeded 70%. Mexiletine (300 mg/day) was 58.5% effective in positive type patients and 33.3% effective in nonpositive type patients, with a total efficacy of 45.8%. Disopyramide was effective in 50% of total cases with 44.4% in positive type patients and 54.5% in nonpositive type patients. However, the efficacy of these drugs on the 2 different types of VPCs was the same statistically. The findings strikingly contrasted those obtained with diltiazem and atenolol, which predominantly suppressed VPCs of the positive type which share similar characteristics with a triggered activity in vitro. We conclude that the mode of action of class I antiarrhythmics on the VPCs differs from that of class II or IV antiarrhythmics, as viewed from the VPC-HR relationship, and that the difference probably comes from the different arrhythmogenesis for positive and nonpositive types of VPCs, in addition to the different electrophysiological actions of mexiletine and disopyramide.


Assembly of 100 nm periodic fibrils (type VI collagen) in human infant corneal stroma. An experimental model for the age-related changes in the extracellular matrix of the human cornea was developed. Human infant corneal stroma in which no long-spacing collagen occurred naturally was treated with 20 mM adenosine triphosphate (ATP). The ATP-treated cornea was observed and compared with nontreated control specimens by electron microscopy. Numerous 100 nm periodic fibrils which resembled long-spacing collagen were formed by the treatment. These experimentally formed fibrils appeared to attract and aggregate the collagen fibrils with D-periodicity, which indicated a connection between type VI and type I collagen. By ruthenium red staining, the cross-bands of the 100 nm periodic fibrils were positively stained, indicating that glycosaminoglycans or proteoglycans were involved in the formation of these fibrils.


Effects and distribution of intravitreally or subretinally injected silicone oil identified in rabbit retina using osmium tetroxide method. Silicone oil was injected into the eyes of 28 rabbits. Utilizing the specific affinity of osmium tetroxide for lipids, the pathological features and histochemical localization of silicone oil within the retina were studied. This osmium tetroxide technique makes it possible to identify the presence of silicone oil particles in the ocular tissues. It was demonstrated that in eyes with silicone oil injected into the vitreous cavity or subretinal space, silicone oil was detected in the inner retinal layer and in phagocytes in the vitreous cavity but not in the outer retinal layer, retinal pigment epithelium and choroid. In addition, prominent subretinal and epiretinal proliferation with abundant phagocytes containing silicone oil particles were noted in the eyes with subretinal injection of silicone oil through an artificial retinal break. This study suggests that when injected subretinally silicone oil induces excessive subretinal and epiretinal proliferation.


[Landau-Kleffner syndrome].

[Landau-Kleffner syndrome]. The Landau-Kleffner syndrome is characterized by the combination of acquired aphasia and epilepsy. Disturbances appear at an age between 2 and 7 years. The prognosis of epilepsy is favourable, but the outcome of aphasia is different. A 15-year old boy is described as one who is totally unable to communicate through speech even 11 years after onset of aphasia. Diagnostics, aetiology, pathophysiology and new therapeutic possibilities of the syndrome are discussed.


[The EEG of patients with tetralogy of Fallot with and without hypoxemic seizures]. Electroencephalograms were recorded of 135 children with TOF. They were normal in 51%, slightly disturbed in one-third, and severe disorders were seen in 16% of patients. Although not statistically significant, cyanotic spells tend to be associated with more severe impairment of the bioelectrical brain function. As far as patients develop additional neurological complications, these result in severe electro-encephalographic changes. Thus, electroencephalographic findings suggest early corrective surgery.


Castration-like effects on the human prostate of a 5 alpha-reductase inhibitor, finasteride. Epidemiological studies strongly support the contention that surgical castration prior to age forty prevents both benign prostatic hypertrophy (BPH) and prostate cancer. 5 alpha-Reductase deficiency in humans, an experiment of nature, is an uncommon genetically transmitted disorder in which prostate size remains very small throughout adult life. A 5 alpha-reductase inhibitor, finasteride, has recently been shown in double-blind, placebo-controlled trials in patients with BPH to statistically decrease prostate size and improve clinical symptoms in comparison to placebo controls. In the untreated BPH prostate, tissue levels of dihydrotestosterone (DHT) and testosterone (T) averaged 4.2 and 0.2 ng/g, respectively. Following one week of finasteride therapy, T levels rose to a mean of 1.32 ng/g while DHT levels decreased to 0.62 ng/g. These values contrast with values in prostate tissue from surgical castrates in which DHT and T values average 1.14 ng/g and 0.1 ng/g, respectively. If we use the relative binding affinity of T and DHT to the androgen receptor as a criterion of biological androgen potency, T would appear to be one-fourth as potent as DHT. Using this 1:4 ratio to convert prostatic T to a biologically equivalent amount of prostatic DHT, the total biologically active DHT equivalent in the prostate following one week of finasteride averages 0.95 ng/g compared to a mean of 1.14 ng/g in surgical castrates.(ABSTRACT TRUNCATED AT 250 WORDS)


Chemoprevention strategies for prostate cancer: the role of 5 alpha-reductase inhibitors. Prostate cancer is a major health problem for the aging male population. Despite hormonal dependence, the inevitable emergence of androgen insensitive tumors, which have a dismal prognosis, highlights the need to develop prevention strategies such as chemoprevention. An acceptable agent must interfere with either the process of carcinogenesis or tumor growth, and have minimal toxicity. In clinical studies, 5 alpha-reductase inhibitors have been shown to suppress serum and intraprostatic levels of dihydrotestosterone, an important promoter of prostate cancer, leading to reduction in prostate size and suppression of glandular cell activity as measured by prostate specific antigen secretion. In addition, 5 alpha-reductase inhibitors have demonstrated an excellent safety profile and tolerability in 12 month controlled clinical trials. No significant metabolic effects have been observed in gonadotropin secretion, spermatogenesis, serum lipids or glucose tolerance. The efficacy and safety of 5 alpha-reductase inhibitors in studies to date, combined with the androgen dependence of tumor production, strongly supports investigating their use for chemoprevention of prostate cancer.


Prostate-specific antigen and diagnosing early malignancies of the prostate. Prostate-specific antigen is a kallikrein-like serine protease that is produced exclusively by the epithelial cells of all types of prostatic tissue, benign and malignant. Physiologically, it is present in the seminal fluid at high concentration and functions to cleave the high molecular weight protein responsible for the seminal coagulum into smaller polypeptides. This action results in liquefaction of the coagulum. Prostate-specific antigen is also present in the serum and can be measured reliably by several different assays. Although the protein is prostate-specific, it is not prostate-cancer-specific. As a result, benign conditions such as benign prostatic hyperplasia, prostatitis and infarction, as well as prostatic intraepithelial neoplasia, can be associated with elevated serum levels of prostate-specific antigen. Approximately 25% of men with benign prostatic hyperplasia have an elevated serum value of prostate-specific antigen, whereas 35% to 40% of patients with organ-confined prostate cancer have a level within the reference range. Prostate-specific antigen can identify some cancers not detectable by digital rectal examination; alternatively, this examination can identify cancers not detectable from the serum prostate-specific antigen concentration. Thus, the most complete evaluation of the prostate gland is achieved when both the prostate-specific antigen value and the digital rectal examination are used. The density and the rate of change of serum prostate-specific antigen are new concepts to improve the ability of prostate-specific antigen to detect early prostate cancer. Preliminary results are encouraging, but additional studies are required to determine the true usefulness of these new variables. Thus, in 1992, determination of the prostate-specific antigen value is a valuable new tool for the practicing physician and will be instrumental in our campaign to diagnose clinically significant prostate cancer at an early, curable stage.


Quantitative morphometric analysis of the microcirculation in prostate carcinoma. Many neoplasms have been shown to induce capillary neovascularization and this may correlate with aggressive behavior. We investigated the phenomenon of neovascularity in benign and malignant prostatic tissue. Microvessel profiles and tissue sections were visualized by antibodies to Factor VIII and standard immunohistochemical techniques, and quantified utilizing the Optimas computerized image analysis system. Microvessel density was compared in benign and cancerous portions of 15 radical prostatectomy specimens. Fourteen of 15 cases demonstrated significantly higher vascular density in the area of carcinoma as compared with benign tissue (ratio = 2.02, p < 0.001). Distribution of microvessels within malignancy was random, whereas it was restricted primarily to the periglandular space in benign tissue. Among 20 men undergoing radical retropubic prostatectomy, there was a correlation between the vessel density and the pathologic stage. No patient with organ-confined carcinoma or cancer penetrating (but not perforating) the capsule had microvessel density greater than 156 microvessels/mm2. In contrast, six of 15 men with more advanced pathologic stage exceeded this arbitrary threshold. These data demonstrate both increased vascularity of prostatic carcinoma as compared with benign tissue, and a further correlation between pathologic stage and vascularity. Microvessel density may be useful as a prognostic indicator.


The role of digital rectal examination, transrectal ultrasound, and prostate specific antigen for the detection of confined and clinically relevant prostate cancer. In a study population, can digital rectal examination (DRE), transrectal ultrasound (TRUS), and prostate specific antigen (PSA) (monoclonal) effectively detect the majority of clinically relevant cancer? If this is possible, the remaining patients could then be considered for chemopreventive protocols. The American Cancer Society/National Prostate Cancer Detection Project (ACS/NPCDP) had a cancer detection rate of 2.4% for its initial year utilizing PSA, DRE and TRUS. TRUS and PSA detected 73% more cancer than DRE alone. TRUS detected a greater percentage of cancers than DRE (85% vs. 64%). PSA was > or = 4 ng/ml for 66% of prostate cancer patients; 11% of cancer patients had PSA < 2 ng/ml. PSA decision levels based on gland volume detected a subgroup at the 95th percentile that had a nine-fold increased risk for cancer. In a separate study differentiating benign prostatic hypertrophy (BPH) and cancer, we found 0.12 +/- 0.13 ng/ml/gm for serum PSA (sPSA)/gm BPH. This study proved that predicted PSA (pPSA) = gland volume x 0.12; this equation also functioned at the 95th percentile for any individual patient. Individual patient assessment: 1. Entry level PSA = 2 ng/ml. 2. Those patients with PSA > 2 ng/ml have TRUS determination of gland volume (performed by technician). 3. pPSA = gland volume x 0.12. If sPSA > pPSA then: 4. (sPSA-pPSA)/2 = predicted volume (cc) of cancer; 5. 3 square root of volume of cancer = mean diameter (cm) of cancer. Thus, these results should detect the majority of clinically relevant cancer (> 0.5 cc). PSA combined with TRUS and DRE can identify high risk groups for cancer.


A retrospective analysis of immunohistochemical staining in identification of poorly differentiated round cell and spindle cell tumors--results, reagents and costs. Immunohistochemistry has rapidly established a significant role in diagnostic pathology. We use immunohistochemistry as an adjunct to morphological diagnosis and employ a "panel approach" to the classification of poorly differentiated tumors. This retrospective analysis was conducted to examine the efficacy of such an approach, using, as an example, the two most common categories of poorly differentiated tumors, namely, the poorly differentiated round cell tumors and spindle cell tumors. Five hundred and fifty-seven consecutive cases of such tumors, collected over a two-yr period, had been subjected to immunohistochemical staining because specific or definitive categorization of the tumor was not possible on the basis of the examination of hematoxylin and eosin stained sections. The clinical history, gross and microscopic findings, as well as the results of immunohistochemical stains were analyzed. Immunohistochemistry allowed a definitive diagnosis to be assigned in 420 cases (75.4%). It was particularly useful in identifying malignant melanoma of both epithelioid and spindle types and distinguished between melanoma, lymphoma and poorly differentiated carcinoma in 126 cases of such lesions occurring in adult lymph nodes. It was also useful in identifying tumors in small biopsies where poor cytomorphological preservation or small size precluded accurate categorization. The application of appropriately chosen panels of antibodies tailored to a narrow list of differential diagnoses helped to identify myogenous, vascular, nerve sheath and fibrocystic lesions among the group of spindle cell tumors. Immunohistochemistry provided definitive diagnoses in 70% of round cell tumors and 92% of spindle cell tumors.(ABSTRACT TRUNCATED AT 250 WORDS)


Rare earth metal ions for unprecedentedly fast RNA hydrolysis. Adenylyl(3'-5')adenosine (ApA) and uridyl(3'-5')uridine (UpU) are hydrolyzed at unprecedentedly large rates by rare earth metal ions at pH 8, 30 degrees C. With 0.01 M Tm(III), the half-lives are 10 min and 51 min, respectively. Potentiality of these ions as catalytic center of artificial ribonuclease is proposed.


Stability difference between DNA and RNA mini-hairpins containing two G-C pairs. The deoxyribonucleotide fragments, d(GCGAAAGC) and d(GCGAAGC) form extraordinarily stable mini-hairpin structures containing only two G-C pairs. In contrast, the corresponding ribonucleotide fragments, r(GCGAAAGC) and r(GCGAAGC) are not so stable, although they also form mini-hairpin structures. The stability difference between these DNA and RNA mini-hairpins was examined by NMR studies and the molecular mechanics calculations, and by comparing with the stability of several sequence variants. Their stability was deduced to vary delicately depending on helical patterns formed by DNA (B form) and RNA (A form) structures.


Properties of hammerhead-type RNA enzyme derivatives which contain a G-to-I replacement in the loop region. Three hammerhead-type ribozyme derivatives containing a G-to-I replacement in the loop regions were chemically synthesized and their properties were examined. All three complexes showed a markedly reduced RNA-cleaving activity. NMR studies suggested that the complex with mutation at the third guanosine of the longer internal loop region has the greatest influence on the conformation of the loop regions.


15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis. Escherichia coli tRNAs were labeled with stable isotope 15N in vivo. Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed. In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other. Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs. Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs. By using a newly constructed E. coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.


In vitro study of E. coli tRNA identity elements. Various tRNA transcripts were constructed to study the identity elements of E. coli tRNAs (Arg, Lys, Ala, Trp, Thr, Gly, Ser, Asn, Cys, His). Anticodon are involved in the identity elements in these tRNA species except the case of tRNA(Ala) and tRNA(Ser). Especially, the second and third positions of the anticodon are the recognition sites of E. coli tRNA(Arg), tRNA(Lys) and tRNA(Thr) for their cognate aminoacyl-tRNA synthetases. Discriminator base is an identity determinant of the above examined tRNAs except tRNA(Thr) and tRNA(Ser). In some cases, acceptor stem (Thr, Gly, His) and variable pocket (Arg, Ala) are considered to be the recognition elements.


Evolution from semi-tRNA to tRNAs, rRNAs and an early peptide-synthesizing RNA molecule. tRNA was found to have emerged by tandem duplication of a ca. 37-base RNA ("semi-tRNA"). The most ancestral anticodon was "(5')CCA(3')". The peptidyltransferase (PT) region of 23S rRNA was concluded to be a 5S rRNA homologue. Important evidences were obtained for elucidating that the RNA transcript from the Bacillus subtilis (BSU) trrnD operon is a relic of an early peptide-synthesizing ribozyme.


Substitution of non-catalytic stem and loop regions of hammerhead ribozyme with DNA counterparts only increases KM without sacrificing the catalytic step (kcat): a way to improve substrate-specificity. In elucidating structure-function relationships and stabilizing ribozymes in vivo, several chimeric RNA/DNA ribozymes and substrates were chemically synthesized. Measurements of kinetic parameters revealed that the maximally deoxyribonucleotide-substituted ribozyme (DRDRD32) gained the highest catalytic activity reaching the kcat value of > 10 min-1, the highest value ever reported for hammerhead-type ribozymes. Since these chimeric ribozymes are more stable than the wild-type all-RNA ribozymes in vivo and they also possess higher substrate-specificity, they are considered to be better candidates for antiviral therapeutic agents.


Inhibitory effects of 2'-deoxy-5-styryluridine 5'-triphosphate analogs on retroviral reverse transcriptase and eukaryotic DNA polymerases. Some sugar modified analogs of 2'-deoxy-E-5-styryluridine triphosphates were synthesized and examined for their inhibitory effects on eukaryotic DNA polymerases and HIV-1 reverse transcriptase. Among these compounds, 3'-azido-2',3'-dideoxy-E-5-styrylUTP (6) and 2',3'-dideoxy-E-5-styrylUTP (7) showed remarkable inhibitory effects on reverse transcriptase. The mode of action and the influence of the substituent at C-5 of 2',3'-dideoxyUTP analogs will be described.


The 6-azauridine analogues possessing sedative and hypnotic effects. Pharmacological effects of 6-azauridine (4) analogues were evaluated using hypnotic activity, pentobarbital (PB)-induced sleep prolongation and locomotor activity as indices. Compound 4, N3-benzyl- (10), N3-o-xylyl- (11), N3-m-xylyl- (12), N3-p-xylyl- (13), N3-alpha-phenylethyl-substituted 6-azauridine (14) exhibited hypnotic activity and PB-induced sleep prolongation, whereas N3-alkyl substituted analogues (methyl-, ethyl-, n-propyl-, n-butyl- and allyl-substitution) did not. Compound 4 and xylyl analogues (11-13) significantly decreased locomotor activity of mice by i.c.v. injection and produced motor incoordination. The results indicate that 4 and its benzyl related analogues, but not alkyl analogues have depressant effects on the central nervous system (CNS).


RNA-RNA and RNA-DNA ligation with the sTobRV(+) hammerhead ribozyme. The sTobRV(+) ribozyme consists of a small catalytic domain and two wing sequences(1). By changing its wing sequences, the ribozyme can cleave many different RNAs in a site-specific manner, functioning as an RNA restriction enzyme(1). Although relatively strong ligase activity is known to be associated with sTobRV(+) RNA(2,3), the sTobRV(+) ribozyme itself has been claimed to have no ligase activity. Here, we show the evidence that the sTobRV(+) ribozyme has the ability to rejoin its digestion products at low temperatures such as 4 degrees C. In contrast, little or no ligation product can be produced at 50 degrees C, the temperature giving the maximum digestion activity. The ligation reaction requires Mg++ ion. The first substrate (P1, see Fig.1), possessing 2',3' cyclic phosphate, must be RNA, but the second substrate (P2), required to have 5'OH, can be replaced by DNA counterparts, equal to or longer than dimer, thus making it possible to generate RNA-DNA chimeric molecules. We also show the resultant RNA-DNA chimera to be digestable by the sTobRV(+) ribozyme. RNase digestion indicates the phosphodiester linkage thus generated to be exclusively 3'-5'.


Application of L-DNA to the study of the specific DNA recognition mechanism of bleomycin. The hexadeoxynucleotide analog, L-d(CGCGCG) composed of L-deoxyribose was synthesized and clearly shown to have the same conformation and dynamic properties with natural D-d(CGCGCG) except for chirality with CD spectra. This unnatural hexanucleotide was not cleaved by bleomycin, an antitumor DNA cleaving drug, but was able to bind to the DNA binding domain of bleomycin to a similar extent with the natural one. These results strongly suggest the importance of the other moiety than the DNA binding domain for the specific DNA recognition of bleomycin. Thus, L-oligonucleotides are useful for the study of DNA-drug interactions.


Does RNA hydrolysis involve rate-limiting formation of the pentacoordinate intermediate or rate-limiting decomposition? Phenyl ester of adenosine 3'-phosphate as a novel probe. The rate-limiting step of enzymatic and non-enzymatic hydrolysis of RNA has been experimentally determined by use of phenyl ester of adenosine 3'-phosphate as a novel probe. Alkaline hydrolysis of RNA involves rate-limiting decomposition of a pentacoordinate intermediate, and any catalyst, either enzymatic or non-enzymatic, should promote the decomposition step.


Cross-ligation and exchange reaction of RNA catalyzed by hairpin ribozymes. The catalytic domain in the minus strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) assumes a hairpin-like secondary structure. This ribozyme catalyzes a cross-ligation reaction between substrate RNAs of different lengths. We constructed ribozymes to probe the activities of ligation and RNA fragment exchange.


OligoDNA-oligoamine hybrid for facile and sequence selective hydrolysis of RNA. Oligoamines as catalytic sites for RNA hydrolysis are attached to 5'-end of [dT]16 using urethane bond. The [dT]16-pentaethylenehexamine hybrid promptly hydrolyzes poly[A] at pH 8, 50 degrees C. Definite importance of the complexation between the poly[A] and the [dT]16 moiety is confirmed by a marginal hydrolysis of poly[C]. Hydrolytic catalysis is ascribed to intramolecular acid-base cooperation of two or more amino residues in the oligoamines.


High-performance separation of polynucleotides by capillary gel electrophoresis. Capillary gel electrophoresis was applied to the high speed separation of DNA and RNA. Factors affecting resolution and speed were optimized for the single base resolution of polynucleotides. Polynucleotides up to 350 bases were completely resolved within 38 min under optimum conditions.


Characterization of four-stranded DNA and RNA fragments. We have investigated the structures formed by deoxyribo- and ribooligonucleotides containing guanine-rich sequences. Inter- and intrastrand guanine tetrads are not only more stable in the presence of K+ than they are in the presence of Na+, but also more compact.


Kinetic studies of the interaction of synthetic RNAs with quinacrine and its analogs. Temperature-jump relaxation method has been used to study the interaction of synthetic RNAs with quinacrine (QAC) and its analog. Two relaxation times were observed. The dependence of relaxation times on the RNA concentration and optical properties of the RNA-dye complexes suggests that (1) QAC binds to poly-(rA).poly(rU) through two bimolecular reactions including isomerization from one complex form to another and (2) the 2-methoxy group of the acridine ring plays a significant role in the isomerization.


Conformational features of the four successive non-Watson-Crick base pairs in RNA duplex. A tridecaribonucleotide, r(UGAGCUUCGGCUC) doesn't form hairpin or interior loop and forms a double helix of 12 base pairs including the four successive nonstandard base pairs, U.G-U.C-C.U-G.U, in the crystal. Non-Watson-Crick base pairs, G.U and U.C are nicely incorporated in RNA duplex maintaining the regular A-form backbone. There exist the good overlapping between base pairings, U.G and U.C, so as to stabilize the nonstandard base pair track. Hydrogen bond networks involving water molecules in the major and minor grooves to stabilize this mismatch base pairing array, are observed and its conformational features are described.


Fluorescence resonance energy transfer between specific-labeled sites on DNA. Fluorescence resonance energy transfer on DNA has been studied for the estimation of distances between specific sites. Two kind of fluorophores, donor and acceptor, were incorporated on double-stranded DNA via phosphorothioate linkage (Sp, Rp, or racemic mixture). The thermal stability of labeled DNA's was slightly dependent on the stereochemical orientation of fluorophore, however all of the duplex structures were stable under the conditions for fluorescence study. The distances between donor and acceptor fluorophores, estimated from fluorescence energy transfer, generally agreed with the expected distance in a B-type DNA for the limiting distance.


Spectroscopic studies of the interaction of synthetic RNAs with quinacrine and its analogs. The interaction of synthetic RNAs with quinacrine (QAC) and its analogs has been studied by absorption, fluorescence and circular dichroism (CD) measurements. The results indicate that the 2-methoxy group of the acridine ring plays an important role in the appearance of the new absorption band upon binding of QAC to poly-(rA).poly(rU).


Origin of sequence specific cleavage of DNA by bleomycins. A new DNA photocleaving agent which contains bleomycin A2's DNA binding portion and its mono- and terthiazole analogues have been designed and synthesized, and their DNA binding mode and cleavage base specificity have been studied. The photoactive p-nitrobenzoyl group attached at the end of molecules cleaves DNA on UV irradiation. All the oligo-thiazole compounds exhibited high sequence specificity in DNA scission. The bithiazole derivative did not cleave DNA at or near 5'-GpT-3' or 5'-GpC-3' as expected from widely believed DNA binding mechanism of the antibiotics.


[Lipid peroxidation and proteinase inhibitors in experimental osteoarticular tuberculosis]. Changes in lipid peroxidation parameters (LP), antioxidant system and proteinase inhibitors were studied at different evolutionary stages of experimental osteoarticular tuberculosis in 30 rabbits. All stages of the process were characterized by LP activation which in animals of all groups (except rabbits having the acute stage of arthritis) is accompanied by compensatory increase of superoxide dismutase and ceruloplasmin activity. Deficiency of superoxide dismutase found in the acute stage of arthritis with a growth of LP activity shows its inadequate production at a given stage of articular inflammation. Decrease in alpha 1-proteinase inhibitor activity at the stage of osteitis with reactive synovitis can be caused by the influence of LP products. The importance of this mechanism in the inhibition of alpha 1-proteinase inhibitor, together with reduction in its production at the acute stage of arthritis, cannot be ignored. The results obtained make it possible to recommend further study of the perspectives of antioxidants use in osteoarticular tuberculosis.


Therapeutic imagery enhanced by hypnosis. This paper has reviewed the history of using imagery as a powerful change and healing agent in humans. It has been a rather underused technique in the practice of Western medicine and psychiatry. I hope that the specific examples and techniques described herein will stimulate and motivate the reader to adopt them in their clinical practice and creatively develop new strategies and techniques applicable in other fields such as sports, the arts, education, and the human life cycle.


Autoradiographic localization of 125I-galanin binding sites in the blowfly brain. The localization of porcine galanin (pGAL) binding sites in the brain of the blowfly Phormia terraenovae was investigated by autoradiography using the following radioiodinated ligands: pGAL 1-29 (two isoforms), pGAL 15-29 and rat (r) GAL 1-29. The different porcine radioligands bound specifically with the following intensity: 125I-[Tyr26]-pGAL15-29 > > 125I-[Tyr26]-pGAL1-29 > > 125I-[Tyr9]-pGAL1-29. With rat galanin 125I-[Tyr9]-rGAL1-29 no specific binding could be shown. In addition, displacement of 125I-[Tyr26]-pGAL1-29 was tested with pGAL 1-29, pGAL 1-22 and pGAL 15-29 (at 0.1 nM-1 microM). A gradual displacement was achieved with increasing concentrations of pGAL 1-29 and pGAL15-29, whereas no displacement with pGAL 1-22 was detected. The results indicate that the C-terminal portion of pGAL is important for binding in the blowfly. The pGAL binding sites were localized in synaptic neuropils of the central body, the antennal lobes, the optic lobes, the pars intercerebralis and the subesophageal ganglion, all of which contain GAL-like immunoreactive neural processes.


Expression of decay-accelerating factor on synovial lining cells in inflammatory and degenerative arthritides. The decay-accelerating factor (DAF) is a complement regulatory cell surface protein that protects cells from complement-mediated lysis. We analysed synovial tissue biopsies from patients with chronic arthritides for the presence of DAF using immunohistochemistry. DAF was expressed in the synovial lining cell layer both in rheumatoid arthritis (RA) and in osteoarthritis (OA). DAF was also on vascular endothelial cells of synovial tissue. A significant correlation was found between the expression of DAF and of HLA-DR in the lining layer, suggesting that DAF may be induced during a local inflammatory response. In addition, C5b-9 terminal complement complexes were found in several DAF-positive cases, suggesting that complement activation might, in itself, induce DAF expression. We propose that the occurrence of DAF may represent a physiological mechanism for local complement regulation in synovial tissue.


Expression of a novel family of epitopes on small intestinal mucins in colorectal cancers, adjacent and remote mucosa. We describe the production, immunochemical and immunohistochemical characterization of monoclonal antibodies (MAbs) raised against the oncofetal small intestinal mucin antigen (SIMA). Four MAbs, reacting with distinct neuraminidase-sensitive SIMA epitopes, were shown to define a novel differentiation-associated relationship of SIMA epitopes within the normal small intestinal villus. Using Swiss rolls of 15 entire colorectal cancer resections, inappropriate expression of SIMA epitopes was detected in all cancers, in adjacent transitional mucosa and remote morphologically normal mucosa, extending as far as resection margins (73%). SIMA expression, whether preexisting or reactive to the tumor, may predispose to malignant change and tumor recurrence.


Tolerance induction to an alloepitope involves antibodies interacting with the T cells activated by the alloepitope. Hyperimmunization of BALB/c mothers with Ab1 (BALB/c anti-C57BL/6) antibody can alter the T-cell repertoire of the syngeneic offspring called BALB/c-p-Ab1. The latter are rendered tolerant to specific alloantigens and are therefore resistant to graft-versus-host disease (GVHD), when challenged 24-48 hours after birth with allogeneic spleen cells. These offspring appear to express cell structures recognized by hyperimmunized mothers and which are presumably the T-cell counterparts of Ab2. Our data suggest that tolerance induction in our mouse model may be modulated by an idiotype network acting during foetal life. The factor modifying the foetal cell repertoire is identified as Ab2 (BALB/c anti-Ab1) antibody transplacentally transmitted from mother to foetus, which seems to carry an "image" of the GVHD-inducing alloepitope and can induce a "GVHD-like" syndrome when passively injected intraperitoneally in normal BALB/c newborn mice.


Expression of MUC1 and MUC2 mucins by human tumor cell lines. The secretion and nature of mucins produced from a panel of recently available new gastric and colon carcinoma cell lines (LIM1839, LIM1215, LIM1863, LIM1899, LIM2099, LIM2405, LIM2408, LIM2412, LIM2463), as well as other colon (LS174T, HT29, HT29-SB, COLO533, COLO206), breast (T47D, MCF-7, BT20, ZR75-1) and ovarian (COLO316) tumor cell lines, was investigated. ELISA and Western blotting of the culture supernatants with novel anti-MUC1 and anti-MUC2 monoclonal antibodies (MAbs) specific for mucin core proteins showed their secretion by most of these cell lines. In addition, mucins produced by these cell lines expressed the tumor-associated carbohydrate detected by MAb 3E1.2 (glycolylsialyl-Tn, mammary serum antigen or MSA) and the Tn or T antigens reactive with lectin SSA-M. SSA-M detected MUC1 or MUC2 captured by MAbs BC2 or CCP58, while 3E1.2 only detected MUC1-associated carbohydrate, indicating that the MAb may react with a conformationally dependent epitope, or that the sialyl/glycolyl-transferases involved in MSA production may be sequence specific. In addition, the BC2/SSA-M and CCP58/SSA-M assays detected mucins in some samples which were not detected by BC2/BC2 or CCP58/CCP58 dual determinant assays, indicating that this format may be more appropriate for the detection of tumor-associated mucins in body fluids. These new cell lines and assays should be of use in the investigation of mucin core proteins, particularly LIM2463 and LIM1839 which express significant quantities of both MUC1 and MUC2.


Determination of optimum cutoff levels of plasma des-gamma-carboxy prothrombin and serum alpha-fetoprotein for the diagnosis of hepatocellular carcinoma using receiver operating characteristic curves. Optimum cutoff levels for plasma des-gamma-carboxy (abnormal) prothrombin (DCP) and serum alpha-fetoprotein (AFP) were determined by analyzing receiver operating characteristic (ROC) curves to discriminate between hepatocellular carcinoma (HCC) and benign hepatic conditions. Plasma DCP levels in 200 patients with HCC and 197 control patients with benign liver diseases were measured by an enzyme immunoassay with anti-DCP monoclonal antibodies, while serum AFP levels for both groups were measured by radioimmunoassay. From ROC curves and tangential lines with a slope of 1.0, the cutoff levels of DCP and AFP were determined to be 0.11 AU/ml and 150 ng/ml, respectively. Lowered cutoff levels of DCP did not improve the sensitivity, in contrast to the increased sensitivity obtained by lowering the specificity of AFP. The sensitivities and specificities determined in this study were close to the currently used values of 0.1 AU/ml for DCP and 200 ng/ml for AFP, justifying these cutoff levels for the differentiation of benign and malignant liver diseases.


Immunohistochemical detection of double-stranded-RNA-dependent protein kinase (p68) with a novel monoclonal antibody TJ4C4. A case report of an AIDS-associated Kaposi's sarcoma treated with alpha-interferon. Double-stranded RNA (dsRNA)-dependent protein kinase (p68) has been shown to be induced by alpha-interferon (IFN-alpha) in mammalian cells. It binds to dsRNA, and is believed to be a factor in the control of both cellular and viral protein synthesis. This report describes the use of a new monoclonal antibody (MAb) TJ4C4, to monitor levels of p68 in a patient with AIDS-associated Kaposi's sarcoma. Using a novel immunoperoxidase/iron staining method, we examined formalin-fixed, paraffin-embedded biopsies prior to, and 4 months after the initiation of IFN therapy. Immunostaining showed low levels (1+ staining) of p68 in the pretreatment tissue, whereas a marked increase (4+ staining) was noted during interferon treatment. This staining suggests an increased level of intracellular p68 expression. This patient has subsequently remained on IFN-alpha therapy and is alive with no evidence of Kaposi's sarcoma, 6 1/2 years after diagnosis. The use of MAb TJ4C4 will greatly facilitate the study of p68 kinase in clinical tissues, and may provide a way to monitor the effects of IFN therapy.


A microcorrosion cast study for analyzing a new method of indirect myocardial revascularization. In order to study the process of angiogenesis a free skeletal muscle flap was transplanted onto the myocardium of the dog. A microvascular anastomosis with the internal mammary artery (IMA) provided the blood flow to the free graft. In 6 dogs, a myocardial infarction of the anterior wall was induced in order to produce a tissue injury. Four weeks later the skeletal muscle flap was transplanted onto the area of infarction in two dogs with its pars anterior in contact with the heart. In two dogs, the muscle was grafted with its pars posterior, i.e. the muscle fascia between the heart and the graft. In two other dogs, the flap was transplanted with its pars anterior in contact with the heart onto a healthy myocardium. Two animals with infarction were left as controls. Eighteen weeks later a microcorrosion cast was prepared from the hearts with the muscle flap. This evidenced neoangiogenesis in the heart only after transplantation of the graft with its pars anterior onto the myocardium whether it was ischemic or not. The intact muscle fascia, though, inhibited sprouting of vessels. In the controls no angiogenic activity was noted. Results are discussed with regard to the probable origin of the neovessels.


[Value of experimental and clinical methods on the effectiveness and side effect profile of anti-arrhythmia agents]. The suppression by antiarrhythmic drugs of ventricular premature beats, pairs and salvos is frequently not associated with a suppression of ventricular tachycardia or fibrillation in the same patient. The evaluation of antiarrhythmic drugs must therefore distinguish between antiectopic, anti-reentry and anti-fibrillatory efficacy. Clinical methods such as Holter monitoring and programmed electrical stimulation which only allow the validation of anti-ectopic or anti-reentry efficacy present a major problem in the evaluation of antiarrhythmic drugs. Anti-fibrillatory drug efficacy cannot be evaluated by clinical methods.


[Combination anti-arrhythmic drug therapy]. Antiarrhythmic treatment with single agents is often ineffective and can be limited by dose-dependent side-effects. Therefore, combinations of antiarrhythmic drugs in smaller and well-tolerated doses are advocated in cases refractory to single antiarrhythmic drugs. Basically, substances belonging to the same electrophysiologic class should not be combined. A combination of beta-adrenoreceptor blockers with class I antiarrhythmic drugs may be effective, mainly in cases in which the arrhythmia is dependent on adrenergic stimulation. As shown in our study, the combination of class III and I B-substances can be useful in some cases, from the electrophysiological and clinical points of view. Among the successful combinations of this type are amiodarone and mexiletine, sotalol and mexiletine, and sotalol and tocainide. In patients refractory to amiodarone alone or to a combination with mexiletine, the combined treatment with amiodarone and class-I-C drugs such as flecainide and encainide prolongs the cycle length of ventricular tachycardia, but does not suppress induction of ventricular tachycardia during programmed stimulation. Combination therapy with amiodarone and encainide is associated with a remarkable incidence of proarrhythmic effects. Nevertheless, a combination of antiarrhythmic drugs improves efficacy of therapy in selected patients. However, a close monitoring is mandatory because of the risk of proarrhythmia.


[Autoantibodies against the beta 1-adrenergic receptor in myocarditis and dilated cardiomyopathy: localization of two epitopes]. Sera of patients with myocarditis and dilated cardiomyopathy contain stimulatory autoantibodies directed specifically against the beta 1-adrenergic receptor. The binding of the antibodies could be localized to either the first or the second extracellular loop of the beta 1-adrenoceptor. In 73% of the cases investigated the antibodies recognized the second extracellular loop. The agonistic effects of the antibodies were abolished by beta-adrenergic antagonists. Furthermore, the antagonists were able to remove the antibodies from their binding sites.


An unusual cause of factitious mineralocorticoid excess. The presence of hypokalaemia in hypertensive patient must prompt a search for increased mineralocorticoid activity. We describe and discuss the observation of a patient with biological markers of hypermineralocorticoidism, despite low plasma and urinary aldosterone levels, and suppressed plasma renin activity. This typical syndrome of apparent mineralocorticoid excess was secondary, in our patient, to prolonged administration of a mineralocorticoid-containing nasal spray.


Alpha-fetoprotein-producing immature mediastinal teratoma showing rapid and massive recurrent growth in an adult. A case of immature mediastinal teratoma in a 43-year-old Japanese man is reported. The tumor, which was multicystic with solid zones and measured 12 x 6 x 8 cm, arose in the anterior mediastinum. The serum alpha-fetoprotein (AFP) level was elevated to 5,114 ng/ml before surgery. Histologically, the solid zones showed an admixture of irregular glands lined by columnar or cuboidal epithelium set in a spindle cell stroma, some foci of primitive neural tissue, and scattered small nests of hepatoid cells. Immunohistochemically, the hepatoid cells and epithelia lining some of the cysts showed a strongly positive reaction for AFP. Eight months after surgery, the patient died of respiratory failure caused by a rapidly growing massive recurrent tumor, which measured 40 x 24 x 13 cm, in the left thoracic cavity. However, the elevated serum AFP level had been decreasing during the course of the recurrence in response to chemotherapy. The recurrent tumor showed remarkable proliferation of loose mesenchymal tissue without primitive neural tissue. These findings suggest that immature mediastinal teratoma in adults is highly malignant, and that non-AFP-producing mesenchymal tissue played a critical role in forming the rapidly growing massive recurrent tumor in the present case.


[Effectiveness of local prostaglandin instillations in tubal pregnancy in relation to preoperative beta-HCG level]. 33 patients with tubal pregnancy and beta-HCG level less than 2500 mIU/ml were treated with local, laparoscopic Prostaglandin-injection. Re-operation was not necessary and beta-HCG fell to undetectable levels in 73% (p = 0.05). Serial beta-HCG correlated poorly with outcome. In patients with an increasing beta-HCG success was limited to 55% (p = 0.59). In contrast patients with falling or stable beta-HCG values had success rates of 85.7% (p = 0.22) and 83.5% (p = 0.30) respectively. Our data suggests there may be a clinical advantage to the use of prostaglandin-injection for the conservative management of early ectopic pregnancy. However the utility of preoperative serial beta-HCG values in predicting success showed a trend, without reaching statistical significance.


[Effect of thyroxine on lung maturation in rabbit fetuses]. The material comprised 11 pregnant rabbits and 95 fetuses stemming from these rabbits. On the 27-th day of pregnancy laparotomy was performed and 1 microgram of thyroxin was administered to the amniotic sac of each fetus in one uterine horn. The fetuses in the second uterine horn, considered as control group, received 0.9% NaCl into their amniotic sac. On the 28-th day cesarean section was carried out, the fluid was collected, the fetuses were taken out, they were weighed and after the lapse of 45 minutes sacrificed by decapitation. The isolated lungs of the fetuses and the amniotic fluid provided the material for further studies. In the amniotic fluid, the content of lecithin and sphingomyelin was determined by thin-layer chromatography. The lungs of fetuses furnished material being used for accomplishing the following types of investigations: determination of mass weight of the wet lungs, and lungs mass after drying, biochemical examination--lecithin and sphingomyelin content in lung homogenates by thin-layer chromatography; histological examination: hematoxylin and eosin stainings by PAS method, argentation by Perdrau method, oil red, toluidine blue stainings as well as electron microscope examination. It has been disclosed that thyroxin administered into the amniotic sac accelerates the fetal lung maturation in rabbits, expressed by higher content of lecithin and by higher ratio of lecithin to sphyngomyelin in fetal lung homogenates at biochemical examinations; it exerts positive effect on the body mass of fetuses and the fetal lung mass, but it fails to influence the lung hydration degree, the sphyngomyelin content in lung homogenates as well as the content of lecithin in the amniotic fluid. The results of histological examination under light microscope have revealed that the lungs of rabbit fetuses in thyroxin group displayed in 70% morphological maturation. The fetal lungs from the control group hardly in 21.1% showed complete maturation. In electron microscopy the fetal lungs in thyroxin group demonstrated more advanced maturation than in the control group, the interalveolar septa were constructed of smaller number of cells, there was a larger number of mature pneumocytes of II type with more abundant lamellar structures, in the alveolar lumen the surfactant appeared markedly more profusely.


Oxidative destruction of biomolecules by gasoline engine exhaust products and detoxifying effects of the three-way catalytic converter. Aqueous solutions of engine exhaust condensation products were derived from cars powered by diesel or four-stroke gasoline engines (with and without three-way catalytic converter). The cars were operated on a static test platform. Samples of the different exhaust solutions accumulated in a Grimmer-type distillation trap (VDI 3872) during standard test programs (Federal Test Procedure) were incubated with important biomolecules. As indicators of reactive oxygen species or oxidative destruction, ascorbic acid, cysteine, glutathione, serum albumin, the enzymes glycerinaldehyde phosphate dehydrogenase and xanthine oxidase, and the oxygen free-radical indicator keto-methylthiobutyrate were used. During and after the incubations, oxygen activation (consumption) and oxidative destruction were determined. Comparison of the oxidative activities of the different types of exhaust condensates clearly showed that the exhaust condensate derived from the four-stroke car equipped with a three-way catalytic converter exhibited by far the lowest oxidative and destructive power.


[Seasonal fluctuations of substance P and vasoactive intestinal peptide concentrations in nasal secretions of patients with nasal allergy to Japanese cedar pollen]. Substance P (SP) and vasoactive intestinal peptide (VIP) concentrations in nasal secretions and plasma from patients with nasal allergy to Japanese cedar pollen and healthy volunteers were measured from Jan to Dec, 1991 using EIA established by us. Simultaneously, the numbers of airborne pollens of Japanese cedar and cypress were counted, and the relation to the SP and VIP concentrations in nasal secretions from the patients with nasal allergy to Japanese cedar pollen was studied. The mean SP concentration in nasal secretions from the patients with nasal allergy to Japanese cedar pollen in the pollination season was 81.9 +/- 48.4 fmol/mg protein, which was significantly higher than that in the non-pollination season (30.4 +/- 14.7 fmol/mg protein) (p < 0.01). Likewise, the mean VIP concentration in nasal secretions from the patients with nasal allergy to Japanese cedar pollen in the pollination season was 14.2 +/- 10.4 fmol/ml protein, which was significantly higher than that in the non-pollination season (4.2 +/- 3.0 fmol/mg protein) (p < 0.01). The SP and VIP concentrations in nasal secretions from the healthy volunteers were not affected by the scattering of pollens. The SP and VIP concentrations in plasma from the patients and the healthy volunteers were not affected by the scattering of pollens.


Modulation of gastric mucosal calcium channel activity by sucralfate. A gastric mucosal calcium channel complex was isolated from solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following reconstitution into phosphatidylcholine vesicles exhibited an active 45Ca2+ uptake and responded to calcium channel activator, BAY K8644, and the antagonist, PN200-110. The uptake of 45Ca2+ was inhibited by an antiulcer agent, sucralfate, which at 100 micrograms/ml evoked maximal inhibitory effect of 52%. The channel complex on epidermal growth factor (EGF) binding in the presence of ATP showed an increase in tyrosine phosphorylation of 55 and 170kDa proteins, and the vesicles containing the phosphorylated channels displayed a 48% greater 45Ca2+ uptake. The phosphorylation process was inhibited by sucralfate, which also interfered with the binding of EGF to calcium channel protein. The results indicate that sucralfate has the ability to protect the cellular integrity from calcium imbalance.


A new approach to the study of ovarian follicles by scanning electron microscopy and ODO maceration. The ultrastructure of the follicle-oocyte complex in rodents and humans was revealed by high resolution scanning electron microscopy (SEM) following the Osmium-DMSO-Osmium maceration method (TANAKA and NAGURO, 1981). In primary follicles, the majority of oocyte organelles such as mitochondria and Golgi complex components are concentrated in a juxtanuclear area. In particular, many spherical mitochondria are oriented all around the nucleus. After maceration of the ooplasm matrix, most of these mitochondria appear intermingled with numerous microtubules (MT) and associated with many Golgi vesicles. Such a nuclear polarization of organelles, essential to the oocyte metabolism, might depend upon a MT activity. MT might guide mitochondria to gather in the perinuclear region and further maintain their close associated to the nuclear envelope. A similar relationship among microtubules, vesicular Golgi complex and mitochondria has been also observed when, in maturing oocytes, these organelles migrate and gather in other areas of the ooplasm. A pattern common only to human developing follicles appears in the occurrence of long microvilli projected from follicle cells deep into the oocyte. These unusual microvilli running within the ooplasm are surrounded by several vesicles of the Golgi complex and endoplasmic reticulum, and often end close to the nucleus. In the antral follicle, the microvilli of corona cells, directed toward the oocyte (after their full exposure through the chemical dissolution of the zona pellucida matrix) are extremely numerous (up to 70/cell), long (up to 7/10 microns) and tortuous. They resemble epididymal stereocilia, may be ramified and possess bulbous tips. In contrast, oocyte microvilli are thin and short.(ABSTRACT TRUNCATED AT 250 WORDS)


Cellular and microvascular changes of the ovarian follicle during folliculogenesis: a scanning electron microscopic study. In order to study the three-dimensional topographic arrangement of the oocyte and zona pellucida, follicular cells and follicle microvasculature, this study applied alkali maceration methods for tissue exposure, the ruthenium red-detergent method for the extracellular matrix visualization, and the vascular corrosion cast technique to rabbits, guinea-pigs and mice ovaries at different stages of follicular development. Macerated samples showed a gradual differentiation of the oocyte surface. This, in primordial follicles, appeared rather smooth, but, with the follicular development, displayed a gradual increase of blebs and microvilli. The latter widely covered the surface of oocytes contained in large or mature follicles. The outer surface of the zona pellucida showed numerous fenestrations, whereas the inner one was smooth. The ruthenium red-detergent method permitted a well detailed view of the filamentous texture of the zona pellucida. The three-dimensional distribution of the contacts between oocyte and neighbouring follicular cells was clearly evaluated in macerated samples. Follicular cells of primary follicles were characterized by their short cytoplasmic processes reaching the oocyte surface. In secondary follicles, these processes issued secondary processes. In larger follicles, the secondary processes of the corona cells were much longer and thinner, and took a tortuous course to reach the oocyte surface, which ran among the numerous oocyte microvilli. This microvillous arrangement greatly increases contact between the oocyte and corona cells, and suggests a coordinated reciprocal control of the activities of both cell types. These data also showed that the spongy and filamentous nature of the zona pellucida is closely dependent upon the temporal differentiation and enormous increase in number of follicular cell projections and their ramifications. Maceration revealed the theca cells surface. In smaller follicles these appeared as fusiform cells which resembled fibroblasts. In larger or mature follicles, many theca cells differentiated to possess morphological features of steroidogenic cells. In addition, these cells delimited a series of intercellular communicating lacunae, continuous with wide pericapillary spaces. The gradual differentiation of the follicle towards a structure having an endocrinal role was further emphasized in vascular corrosion casts. A simple microvascular net made of thin capillaries supplying primary follicles was seen to transform into an elaborate sinusoidal network made of thick permeable capillaries, supplying mature follicles.


Mouse/human chimeric anti-HIV-1 gp120 antibody to the principal neutralizing determinant: tolerability and pharmacokinetics in cynomolgus monkeys, Macaca fascicularis. In preparing for testing a pharmaceutical grade preparation of chimeric (mouse/human) antibody CGP 47,439 in HIV-1 infected individuals, it was administered to Macaca fascicularis (cynomolgus) monkeys to study tolerability, immunogenicity and pharmacokinetics. Four groups of monkeys, three males and three females per group, received respectively four infusions of 0, 1.43, 4.3, and 14.3 mg of CGP 47,439/kg body weight at one-week intervals. The chimeric antibody induced no fever, was tolerated well throughout the 50-day observation period, elicited no tissue damage and no anti-antibody response. The pharmacokinetic profile was similar at all dose levels with a mean T1/2 alpha of 14.2 h (range 11.8-19.3 h) and a mean T1/2 beta of 172.6 h (range 137.2-220.5h). Following four successive antibody infusions serum concentrations of CGP 47,439 increased without reaching a steady state, and its measured concentrations were comparable to the simulated values. Collectively the study has provided safety and pharmacokinetic data that would allow human studies with this antibody in AIDS patients.


ConA induced changes in energy metabolism of rat thymocytes. The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na+K(+)-ATPase, Ca(2+)-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na+K(+)-ATPase with about 20% each of total oxygen consumption, while Ca(2+)-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.


[Beta-adrenoblockers in the reorganizing of the food center of the hypothalamus under the influence of substance P]. An ability of substance P (15 nmol intracerebroventricularly) to transform functional properties of the hypothalamic feeding center manifested in escape reaction in response to threshold electrical stimulation of this motivation center in rabbits. Beta-adrenergic antagonists inderal and obsidan (0.25, 0.5 mg, respectively) injected intravenously were found to restore feeding elicited by electrostimulation of the hypothalamic feeding center. However, escape reaction, elicited from the feeding center under substance P was found unchanged after intravenous administration of 0.25 mg kalipsol. The data obtained suggested an important role of beta-adrenergic mechanisms in substance P transformation of the functional properties of the hypothalamic feeding centre.


[The effect of pentagastrin and substance P on the gastric parietal glandulocytes]. Investigations on dogs show that substance P suppresses pentagastrin gastric acid secretion. This effect is abolished by acetylcholinesterase block by calimin. The results indicate that pentagastrin and substance P participate in cholinergic regulation of gastric acid secretion.


[The dynamics of the extinction of brain cellular biosynthesis at different times after death]. The paper presents experimental data on RNA synthesis in neuronal, glial, endothelial cells of a dying organism. Under certain conditions it is possible to define labelled cells in the majority of the brain cells within post-death hours 1-4. This fact proves that genome is still functioning. Endothelial cells possess higher vital capacity. CNS neurons are capable of synthesizing RNA within the period of up to 4 hours after death of the organism.


Immunogenetics of collagen induced arthritis in mice: a model for human polyarthritis. Collagen induced arthritis is an experimental animal model of inflammatory polyarthropathy that has many features of human rheumatoid arthritis. Type II collagen is the major matrix protein of hyaline cartilage and is a sequestered protein which can be presented as an autoantigen under certain conditions. To induce CIA, type II collagen is injected intradermally with complete Freund's adjuvant. Susceptibility to CIA is dependent on the presence of the trimolecular complex: 1) the arthritogenic epitope on the type II collagen; 2) a class II MHC molecule on the accessory cell presenting the arthritogenic epitope; and 3) T cells expressing specific V beta chains in their TCRs. Complement and other non-MHC background genes also may play a role in susceptibility to CIA. Both cell mediated and humoral immunity are involved in the pathogenesis of CIA. To date immunotherapies that have modulated CIA include use of anti-class Ii antibodies, anti-lymphokines, and monoclonal antibodies directed against specific cellular markers. All of these therapies are able to modulate disease to some extent but lack the specificity and efficacy to make them practical for widespread use in human disease. Most promising, is the use of monoclonal antibodies directed against specific V beta TCR subsets. This is potentially a very specific and effective therapy because it will affect only the cells involved in disease while leaving the host otherwise immunocompetent. Therapies on the horizon include the use of synthetic peptides with sequences homologous to various regions on the TCR, immunotoxins, and superantigens to modulate the immune response and ameliorate disease.(ABSTRACT TRUNCATED AT 250 WORDS)


The school nurse's role: early intervention with preschool children. With the advent of PL99-457, preschoolers are increasingly appearing in the educational system. The school nurse must have an understanding of the basis for early entrance of these children and methods by which they obtain entry. This article focuses on the diagnosis of developmental delay in preschool children and identifies some of the specific roles and functions involving the school nurse.


Heterogenous distribution of peptide-containing nerve fibres within the circular muscle layer of the human pylorus. The distribution of nerve fibres immunoreactive for vasoactive intestinal polypeptide (VIP), substance P (SP), methionine-enkephalin (ENK), calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) within the circular muscle layer was examined histochemically in the human pylorus, adjacent antrum and duodenum. Longitudinal cryostat sections of the pyloric and surrounding regions were stained by an indirect immunofluorescence method, and the total length of each type of peptide-containing fibre per unit sectional area (micron/mm2) was measured using an image-analysing system. The narrow region of the circular muscle layer bordering the submucosa in the pylorus contained a rich supply of VIP, SP, ENK and CGRP immunoreactive fibres; VIP fibres were most prominent with less SP and ENK fibres and moderate amounts of CGRP. These peptide-containing nerve fibres were more dense than in the pyloric circular muscle, the longitudinal muscle layer and also the adjacent muscle layer. NPY-immunoreactive fibres were sparsely distributed throughout the pyloric region. These results suggest that the inner edge of the circular muscle, lying adjacent to the submucosa and densely innervated with peptide-containing fibres, may be a characteristic feature of the human pyloric sphincter.


Protein hydration in aqueous solution.

Protein hydration in aqueous solution. Proton nuclear magnetic resonance was used to study individual molecules of hydration water bound to the protein basic pancreatic trypsin inhibitor (BPTI) and to the nonapeptide oxytocin in aqueous solution. The experimental observations are nuclear Overhauser effects (NOE) between protons of individual amino acid residues of the protein and those of hydration water. These NOEs were recorded by two-dimensional (2D) and three dimensional (3D) NOE spectroscopy (NOESY) in the laboratory frame, and by the corresponding experiments in the rotating frame (ROESY). The studies show that there are two qualitatively different types of hydration sites. Four water molecules in the interior of the BPTI molecule are in identical locations in the crystal structure and in solution. Their NOEs with the protein protons are characterized by large negative cross-relaxation rates sigma NOE, which indicates that the residence times of the water molecules in these hydration sites are longer than ca. 10 ns. Additional experiments with extrinsic shift reagents established an upper limit of 20 ms at 4 degrees C for these residence times. Surface hydration of both the globular protein BPTI and the flexibly disordered polypeptide oxytocin is by water molecules with residence times in the subnanosecond range, as evidenced by small positive sigma NOE values observed for their NOEs with nearby polypeptide protons. Short residence times prevail for all surface hydration sites, independent of whether or not they are occupied by well ordered, X-ray observable water in the protein single crystals.


Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells. The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.


Autoantibody activity and V gene usage by B-cell malignancies. Autoreactive B cells account for a substantial part of the B-cell repertoire. They frequently secrete polyspecific natural autoantibodies, which probably bind with low affinity to the different antigens they recognize and which express germinal genes. The role of this pre-immune repertoire still remains to be defined but it has been suggested that it participates in the elimination of cell breakdown products, serves as a first barrier of defense or acts as a template upon which antigen driven selection and somatic recombinations could induce the emergence of high affinity induced antibodies. The present study, reviews the evidence favouring the idea that this autoreactive B-cell repertoire, which expresses a restricted set of V genes, frequently undergoes malignant transformation. This evidence arises from the study of the autoantibody activity and V gene usage in three different models of B-cell malignancies namely monoclonal immunoglobulins; chronic lymphocytic leukemia; and follicular non-Hodgkin's lymphomas.


Comparison of two methods for concentrating CD34+ cells from patients with acute non-lymphocytic leukemia. The aim of the present study was to compare two different methods for obtaining CD34+ cells from the peripheral blood or the bone marrow of patients with acute non-lymphocytic leukemia (ANLL). Twenty-two samples, obtained from 19 patients, were density cut (Ficoll-Hypaque 1.077) and, after incubation with My10 antibody, separated by panning or by immunomagnetic beads. Immunomagnetic beads provided a significantly better separation than panning, either in terms of concentration of CD34+ cells (85.5 +/- 11.6% vs. 55.7 +/- 25.0%, p = 0.003) or in terms of depletion of CD34+ cells (3.9 +/- 8.0% vs. 30.9 +/- 26.3%, p = 0.008). This was consistent with the virtually complete depletion of colony forming cells (CFC) in the CD34 negative fraction and the recovery of virtually all the CFC in the positive fraction in the samples separated by immunomagnetic beads. In conclusion, separation by immunomagnetic beads can allow collection of nearly pure CD34+ and CD34- cell populations from patients with ANLL, thereby facilitating the study of the biological characteristics of these cell populations. Moreover the method is less time consuming than panning and is not toxic to the CFC.


[Alpha fetoprotein levels in the serum and bronchoalveolar lavage fluid in patients with lung cancer and other diseases of the respiratory system]. Concentration of alpha-fetoprotein (AFP) may be increased in some type of lung cancer. In the study concentration of AFP was evaluated in serum and broncho-alveolar lavage fluid (BALF) in patients with lung cancer. The values of concentration were compared with results obtained from patients with other diseases of the respiratory tract. Examinations were performed in 14 patients with lung cancer, 12 with sarcoidosis, 23 with chronic obstructive bronchitis and 16 with acute bronchitis. Liver pathology was excluded according to biochemical analytical tests. In all patients bronchofibroscopy was performed and BALF was obtained in routine way. Concentration of AFP in serum and BALF was determined by immuno-assay technique. In performed examinations non significant increase of AFP concentration was determined in serum and BALF of patients with lung cancer. However, obtained values were increased twice than in patients with acute bronchitis. Moreover, it was noticed that in patients with chronic obstructive bronchitis the AFP concentration was the highest, especially in the group treated by steroids. The study indicates that evaluation of AFP concentration is out of value in diagnosis and differentiation of lung cancer. It seems to be necessary to continue the examinations for explanation a role of steroids in inflammatory process and increase of AFP concentration.


[Reactivity of pulmonary mast cells and peripheral blood basophils in patients with sarcoidosis and bronchial asthma]. We have studied the sensitivity of pulmonary mast cells and blood basophils in patients with sarcoidosis and bronchial asthma. The cells were induced in vitro with anti-IgE. The histamine releasing from mast cells and basophils was high in both examined groups of patients. The histamine releasing from mast cells in patients with sarcoidosis was 27.8-47.1% and in asthmatic patients 42.8-58.9%. The histamine releasing from basophils was 38.2-57.9% and 41.0-62.1%, respectively. The high sensitivity of mast cells of patients with sarcoidosis may suggest that these cells play an essential role in pathology of this disease.


Comparison of the vasoconstrictor responses induced by endothelin and phorbol 12,13-dibutyrate in bovine cerebral arteries. The vascular effects of endothelin-1 (ET-1) were compared with those elicited by phorbol 12,13-dibutyrate (PDB), an activator of the protein kinase C (PKC), to analyze the involvement of this enzyme on ET-1 responses. PDB and ET-1 caused slow-developing contractions (sustained and transient, respectively), which were reduced by the PKC inhibitor, staurosporine (1 and 10 nM). Only the contractile effects evoked by ET-1 were reduced in Ca-free medium and by the Ca channel antagonist, nifedipine (1 microM), and increased by the Ca channel agonist, BAY K 8644 (10 nM). PDB (10 and 30 nM) preincubation reduced the vasoconstriction elicited by 5-hydroxytryptamine (5-HT; 0.01, 0.1 and 1 microM) in a way dependent on phorbol concentration and preincubation time, whereas ET-1 (1 nM) increased the contractile response to 5-HT (0.1 microM). Furthermore, PDB (0.1 microM) also reduced the responses elicited by ET-1 (30 microM) and vice versa. ET-1 (0.1 microM) induced transient translocation of PKC activity from the cytosol to the membrane, which was less than that produced by PDB (0.1 microM). Electrical stimulation induced [3H]noradrenaline (NA) release, which was increased by PDB (10 and 100 nM) and not affected by ET-1 (10 nM). These results indicate: (1) the responses induced by PDB and ET-1 were independent and dependent on extracellular Ca, respectively; (2) PKC is involved in NA release and 5-HT responses, but mainly in desensitization of these responses, and (3) PKC is activated by ET-1 and is implicated in vascular actions of ET-1, but other mechanisms, such as the activation of ET-1 receptors and opening of dihydropyridine-sensitive Ca channels also appear to be involved.


CRF-like immunoreactivity selectively labels preganglionic sudomotor neurons in cat. Immunohistochemical and neuronal tracing methods were used in cats to determine which type of postganglionic sympathetic neuron is innervated by preganglionic neurons which contain corticotrophin releasing factor-like immunoreactivity (CRF-LI). Preganglionic neurons with CRF-LI have their cell bodies at two restricted levels of the spinal cord and terminate in the stellate and lower lumbar ganglia. CRF-LI terminal baskets in stellate and lumbar ganglia surrounded cell bodies, 96-99% of which showed no tyrosine hydroxylase (TH)-LI (presumptive cholinergic neurons). Calcitonin gene-related peptide (CGRP)-LI was used to label the cholinergic ganglion cells which innervate sweat glands: 96-99% of those were confirmed as lacking TH-LI, while the remainder showed weak staining. Every one of over 6000 CRF-LI terminal baskets counted in 4 stellate and 6 lumbar ganglia was found to surround a cell body with CGRP-LI; conversely, 81-86% of the cell bodies showing CGRP-LI were surrounded by CRF-LI terminal baskets. In 3 cats, the retrograde tracer fluorogold was used to label postganglionic neurons projecting to the paw pads (a population which includes both cholinergic sudomotor neurons and noradrenergic vasoconstrictor neurons). Between 26 and 38% of the retrogradely labelled ganglion cells were surrounded by CRF-LI terminal baskets. We conclude that in cats, preganglionic sympathetic neurons with CRF-LI are sudomotor in function.


Effect of substance P on respiratory rhythm and pre-inspiratory neurons in the ventrolateral structure of rostral medulla oblongata: an in vitro study. The pre-inspiratory (Pre-I) neurons which fire in the pre- and usually also during the post-inspiratory phase are located in the ventrolateral structures of the rostral medulla. They are suggested as primary rhythm generating neurons for respiration. These have been studied in isolated brainstem-spinal cord preparations from newborn 0-5-day-old rats. We have found that application of substance P (SP) enhanced the respiratory rhythm as measured by C4 ventral root and pre-I neuronal activities. Furthermore, the effect of SP was dependent on basal respiratory rate. An increase of the Pre-I and C4 burst rate by SP was clearer when the basal respiratory rhythm was somewhat lower. Moreover, long lasting depression of respiratory rate after the application of the alpha 2-agonist clonidine was reversed by SP. On the other hand, an inhibitory effect appeared in preparations with a higher basal respiratory rate, while the Pre-I burst rate tended to increase during SP perfusion. During chemical synaptic transmission blockade by perfusion with low Ca2+, high Mg2+ solution, a pre-I burst retained or completely blocked was found to be enhanced or reactivated by SP perfusion. The results suggest a direct postsynaptic action of SP, which could strongly stimulate burst generating properties of Pre-I neurons.


Morphine induces an increase in extracellular serotonin in the rat diencephalon. The effect of systemic morphine on extracellular serotonin (5-HT) in the diencephalon of unanesthetized, unrestrained rats was investigated by in vivo microdialysis coupled to high performance liquid chromatography with electrochemical detection. Administration of morphine resulted in a dose dependent increase in extracellular 5-HT. Significant increases were first seen at a dose of 5 mg/kg, and a maximal increase occurred at 10 mg/kg. This increase was blocked by pretreatment with naltrexone, indicating that the effect of morphine on 5-HT was mediated by opiate receptors. Morphine also had a significant effect on extracellular 5-hydroxyindoleacetic acid (5-HIAA), leading to a gradual increase across a range of doses. The effect of morphine on 5-HT was compared to effects of morphine on nociception and catalepsy. Increases in 5-HT and 5-HIAA were first detected at doses that were analgesic but too low to elicit catalepsy. Consistent with many reports that opioids cause an increase in synthesis and turnover of 5-HT in the rat forebrain, the present results provide more direct evidence that 5-HT release is increased after morphine administration.


[A preliminary approach to pathological and biochemical changes of the lungs injured by domestic pingyangmycinum]. Two hundred male mice, weighing 18-24g, were randomized into four groups. A model of lung injury was established in mice by intratracheal administration of a single dose of domestic pingyangmycinum (Bleomycin A5). The dose-effect relationship for different doses of pingyangmycinum (0.05, 0.1, 0.2 mg) on the lung injury, and the change of hydroxyprolin (Hyp), ceruloplasmin (CP), angiotensin converting enzyme (ACE), superoxide dismutase (SOD) in mice were investigated. The results showed that the pathological changes of alveolitis and pulmonary interstitial fibrosis were more severe, and mortality of the mice was increased when pingyangmycinum dose was increased. During 2-6 weeks of this experiment, Hyp, CP, ACE and SOD in serum and Hyp in lung tissue were increased in different degrees. However, the activity of SOD in lung tissue was decreased. The possible mechanism of this lung injury is also discussed.


[Coexpression of intermediate filaments in notochords and chordomas and its significance in the histogenesis and diagnosis of chordoma]. Six chordomas, ten fetal notochords and eight adult notochords were stained for keratin, vimentin, GFAP, desmin, CEA, EMA and s-100 protein. Coexpression of four kinds of intermediate filaments was common in fetal notochords and chordomas. According to the histologic and immunohistochemical characteristics, chordoma may originate from the ectopic remnants of undegenerated notochord during the embryonic stage. The chordoma consists of two cell types; nonvacuolated and physaliphorous cells, and exhibits the potentiality of multidirectional differentiation. The coexpression of different types of intermediate filaments could explain the morphological transformation of chordoma.


[The abnormal cytokeratin expression in HBV-caused hepatitis, early-cirrhotic and cirrhotic livers, its mechanism and significance]. An ABC immunohistochemical study of the expression of cytokeratin (CK19 and CK18) was carried out in 315 cases of HBV-caused hepatitis, early-cirrhotic and cirrhotic livers. It was shown that hepatocytes in 73% of chronic active hepatitis (CAH) (80/110) and 81% of early-cirrhotic and cirrhotic livers (117/144) expressed CK19 (the abnormal CK expression), which could be of help in differentiating CAH from chronic persistent hepatitis, subtype CAH (mild, moderate to severe type) and in determining the activity of early-cirrhotic and cirrhotic livers. The abnormal CK expression was shown to be closely related to the activity of liver disorders. The CK19 expression in hepatocytes had the closest relations with the piece-meal necrosis of hepatocytes, isolation of hepatocytes into groups by connective tissue, and fibrosis. It is suggested that CK19 expression may be one of the local reactions to the piece-meal necrosis of hepatocytes.


[Clinical and pathological features of primary hepatocellular carcinoma in 26 patients survived over 10 years after operation]. The clinical and pathological features of primary hepatocellular carcinoma (PHC) in 26 patients who had survived over 10 years after operation were analyzed. The results were as follows: 84.62% (22 patients) were detected by AFP screening, 80.77% (21 patients) were in clinical stage I, 76.96% (20 patients) were younger than 45 years of age. Serum AFP of all patients turned negative within 2 months after operation. The tumor diameters ranged from 1.2 cm to 15 cm, and solitary tumors accounted for 80.77% (21 patients). All tumors were encapsulated and infiltrated by lymphocytes. Cirrhosis was found only in 9 cases (34.62%). The positive rates of HBsAg and HBcAg were 80% and 15% respectively in hepatic tissues surrounding the carcinoma. These results suggest that good prognosis or long survival is related to early detection, younger age of the patient, solitary tumor, better encapsulation, mild injury of the surrounding liver tissues, and rapid negative turning of AFP after operation.


Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method. The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1 + SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 microM no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.


Effects of calmodulin inhibitors on amylase secretion from rat pancreatic acini. To investigate the role of calmodulin in stimulus-secretion coupling in pancreatic acinar cells, we studied the effects of W-7, a calmodulin inhibitor, and KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II), on amylase secretion from rat pancreatic acini. Calmodulin inhibitor (W-7, 100 microM) and Ca2+/CaM kinase II inhibitor (KN-62, 10 microM) reduced amylase secretion stimulated by cholecystokinin (CCK) or carbachol. W-7 and KN-62 also inhibited amylase secretion stimulated by both calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). To clarify the role of calmodulin in the interaction of intracellular mediators, pancreatic acini were permeabilized with streptolysin O. Following permeabilization, amylase secretion was stimulated by submicromolar free Ca2+, and this Ca(2+)-dependent amylase secretion was enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), TPA or cyclic adenosine 3',5'-monophosphate (cAMP). W-7 and KN-62 had no effects on amylase secretion stimulated by Ca2+ alone, but inhibited the enhancement in Ca(2+)-dependent amylase secretion by GTP gamma S, TPA or cAMP. These data suggest that calmodulin plays an important role in Ca(2+)-dependent amylase secretion from pancreatic acinar cells and in the interaction between Ca2+ and other intracellular messengers.


Sero-surveillance of transmissible hepatitis B & C viruses in asymptomatic HIV infection in haemophilics. In a group of 37 haemophilics, 9 (24.3%) were seropositive for human immunodeficiency virus (HIV), while 9 (24.3%) and 10 (27%) were positive for hepatitis B virus (HBV) and hepatitis C virus (HCV) respectively. Haemophilics who were HIV seropositive had higher prevalence of HBV and HCV. Seropositivity for HIV was more in patients with severe haemophilia A who required frequent factor VIII replacement. The need for long term surveillance of voluntary blood donors for transfusion associated viruses like HIV, HBV and HCV, is emphasized.


Effects of growth hormone administration in addition to gonadotrophins in normally ovulating women and polycystic ovary syndrome (PCO) patients. Follicular fluid sex-steroids, insulin-like growth factor-I (IGF-I), IGF-I binding protein (IGF-I-BP) and epidermal growth factor were investigated in patients with polycystic ovaries and normally ovulating women, following ovulation induction with gonadotrophins or growth hormone plus gonadotrophins. Growth hormone supplementation enhanced the ovarian response to gonadotrophins, and significantly increased follicular fluid IGF-I in both groups, without affecting follicular fluid epidermal growth factor; growth hormone supplementation significantly decreased follicular fluid androstenedione in both groups.