[Correlation between syndrome types of traditional Chinese medicine and peripheral T lymphocytes subsets in Graves' disease].

This paper reports the determined results of OKT3, OKT4, OKT8, ERFC, smIg and CIC, TMCA, TGA in 31 cases of Graves disease and in 20 normal controls. The results showed that the OKT3, OKT4, OKT8, ERFC were significantly lower than those in the normal controls, whereas the smIg was higher than that in normal controls. The difference between the two groups was very significant. Even though the ratio of OKT4/OKT8 showed no significance of both. Typology of Graves disease according to the theory of TCM, all 31 cases were divided into two types: (1) 14 cases of depression of Liver-energy and asthenia of Spleen; (2) 17 cases of deficiency of yin leads to hyperactivity of Fire. The OKT8 and the ratio of OKT4/OKT8 in the latter respectively were lower and higher than those of the former. The difference between the two types was significant (P less than 0.01, P less than 0.05) whereas the positive rates of the CIC, TMCA, TGA also were higher in the deficiency of Yin leads to hyperactivity of Fire than those in the depression of Liver-energy and asthenia of Spleen. After treatment with combined TCM-WM on 31 cases of Graves disease, it was found that the OKT4, OKT8, ERFC were significantly elevated, the smIg was markedly decreased than those without treatment. It was also found that smIg markedly decreased in two types, OKT8, ratio of OKT4/OKT8 in the latter and ERFC in both types all returned to normal. Remainder indexes had no obvious change before and after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


[Qi-xue of traditional Chinese medicine and the electroencephalography energy].

The experiment observed the different changes of EEG energies among 37 patients of neuroasthenic syndrome, 18 patients of anxiety, 28 patients of cerebral apoplexy in recovery stage, 48 cases healthy persons, and 13 healthy persons aged over 60 years. It was found that the changes in EEG energies coincided with the changes in Qi-Xue system. The results showed that analysis of EEG energy could be an objective basis for studying Qi-Xue of traditional Chinese Medicine and the change of EEG energy could be used in judging the condition of Qi-Xue.


[Relation between treatment with traditional Chinese medicine for recurrent aphthous ulcer and human zinc and copper].

The purpose of the study is to explore the relation between general condition of patients with recurrent aphthous ulcer (RAU) and human zinc and copper with TCM conception of the organism as a whole, and adjustment effect of human body zinc and copper by TCM treatment. Results showed that the average value of serum zinc of 75 cases of RAU was on lower level within normal range, serum copper was normal, the rate of copper to zinc was higher than normal value. Analysis using the TCM theory showed serum zinc of patients of deficiency symptom-complex was lower than excessiveness symptom-complex, the rate of copper to zinc of patients of deficiency symptom-complex was higher than normal range. The zinc content of serum and the rate of copper to zinc were different in patients of various symptom-complexes of RAU. The zinc and copper contents of serum were adjusted, the rate of copper to zinc was normalized and the immune function of T-cell increased distinctly by TCM treatment according to an overall differentiation of symptoms and signs. Thus the therapeutic effect of TCM was better than zinc preparation.


[Research of the relation between the type of asthenia of the spleen and kidney and platelet associated antibodies and T-lymphocyte subsets in idiopathic thrombocytopenic purpura].

Idiopathic thrombocytopenic purpura (ITP) is a kind of disease associated with immunity. At present a great quantity of study on ITP has been made on humoral and cellular immunity. But there are few reports about the relationship between the types based on the differential diagnosis of TCM and immune rationale of ITP. In order to deeply explore the relationship between the types based on differential diagnosis of TCM and immune rationale of ITP., the authors measured PAIg and T lymphocyte subsets of 34 ITP patients of asthenia of both Spleen and Kidney. The value of PAIgG increased in both types of Spleen failing to control blood (SFCB) and deficiency of Spleen-yin and Kidney -yin (DSYKY), and the value of PAIgG of the type of DSYKY was significantly higher than that of SFCB (P less than 0.01). OKT3, OKT4/OKT8 of the type of SFCB remarkably decreased (P less than 0.05), OKT4, OKT4/OKT8 of DSYKY also remarkably decreased (P less than 0.001), while OKT8 significantly increased (P less than 0.001). The above results suggested that the type of DSYKY has more serious immune dysfunction than the type of SFCB, and the types of SFCB and DSYKY has close relationship with PAIg T lymphocyte subsets.


[Effect of total saponins of Panax ginseng on hematopoietic progenitor cells in normal human and aplastic anemia patients].

Ginseng was said to be benefit for anemia in TCM. Proliferation effects of total saponins of panax ginseng (TSPG) on hematopoietic progenitor cell in normal individuals and 29 patients with aplastic anemia (AA) were observed by bone marrow culture of BFU-E, CFU-E, CFU-GM in vitro compared with methyltestosterone (MT). The results showed that TSPG might prompt proliferation of normal progenitor cells at the concentration of 20 micrograms/ml. The number of BFU-E, CFU-E and CFU-GM had increased by 37.8 +/- 2.9%, 31.4 +/- 2.9% and 33.3 +/- 4.0% over the controls respectively; furthermore TSPG was still useful to BFU-E, CFU-E growth without Epo in vitro, although the colony numbers were very lower. Otherwise MT was useless to CFU-GM. 14 of the 29 patients with AA who responded to MT showed sensitivity to TSPG in marrow culture (the rising rate of colony formation exceeded 30%), but immune-mediated AA (patient's PBMNC suppressed normal hematopoiesis) and stem cell-decreased AA (few of colony was formed) showed almost no expression for TSPG activity because of immunological suppression system and absence of progenitors.


[Study on the granule of shencao fuzheng kangai].

The clinical effect of the granule of Shencao Fuzheng Kangai had been proved and the animal experiment was carried out. The results showed that: (1) No toxic response was found in acute toxicity test. (2) The granule could prevent WBC from decreasing severely in chemotherapy experiment (P less than 0.01). (3) It was indicated that the granule could improve the phagocytic function of macrophage in carbon clearance experiment (P less than 0.01). (4) It was meant that the granule could inhibit the growing of some solid carcinoma in inoculation experiments.


[Preventive and therapeutic effects of hirudo on incipient acute tubular necrosis in rats].

Male Sprague-Dawley rats, weighing 180-250 g and depleted with water for 16 h, were injected with glycerol (im) to induce acute tubular necrosis, and then divided into groups given blood-activating and stasis-removing drug, Hirudo solution (GH) tap water (GW), verapamil (GV) and none (GSDW) in incipient stage separately. It was observed that levels of BUN increased at 24th and 48th h after administration of glycerol and levels of Bcr increased at 3rd, 24th and 48th h after injecting glycerol in GH were significantly lower than those increased in GW and GSDW (P less than 0.05-P less than 0.001), but roughly similar to those in GV (P greater than 0.05-P greater than 0.5). Renal histopathological damage under light microscope and electron-microscope in GH at 3rd and 24th h after administration of glycerol were also less severe than those in GW and GSDW. The results suggested that Hirudo could exert a preventive and therapeutic effects on incipient acute tubular necrosis induced by glycerol in rats.


[Clinical epidemiological study on risk factors of coronary heart disease in 743 subjects].

This paper sums up the clinical epidemiological investigation data on risk factors (RF) of coronary heart disease (CHD) among 743 office workers, with an average age of 61.0 +/- 8.0. The investigation involved factors relating to history, physical examination, biochemistry, blood rheology and TCM Syndrome Differentiation. According to the results of the computerized single-factor correlation analysis, the incidence of CHD in RF exposed group was obviously higher than that of unexposed one, 65 RF such as hypertension, diabetes, hyperlipemia, smoking, body weight, HDL-C/TC, blood viscosity etc. were recorded. Using multivariate regressive analysis it revealed that hypertension, diabetes, total cholesterol, heavy cigarette smoking, overweight, diastolic pressure, cortisol, TCM senile index, Blood Stasis Syndrome, Qi Stagnation Syndrome, Qi Deficiency Syndrome and Heart Deficiency Syndrome were the main RF. The result concerning RF of Western medicine (WM) was in conformity with that at home and abroad. In addition, some TCM-RF were selected which couldn't be replaced by WM-RF. These indicate that there are TCM-RF and WM-RF in the development of CHD and it is better to adopt the method for preventing and treating CHD with combined TCM-WM. As to TCM-RF of CHD, the authors consider that there are both the factors of Deficiency and Excess, so preventing and treating CHD should aim at reinforcing the Deficiency and reducing the Excess.


[Correlation analysis between plasma atrial natriuretic peptide and cardiac function in blood deficiency syndrome].

The function of ANP in the cardiovascular regulation is very similar with the TCM theory of "the Heart governs blood circulation". Using the method of cardiac impedance to check cardiac output and the method of radioimmunoassay (RIA) to check plasma ANP, the result showed that in the status of Blood Deficiency Syndrome, cardiac function was impaired, there were reduced kinemia and stroke volume, as well as markedly raised plasma ANP and peripheral resistance. The above-mentioned indexes were significantly different from those of normal group (P < 0.01). Using multivariate regression analysis, cardiac output was negatively correlated with the plasma ANP (P < 0.05). 23 cases with Blood Deficiency Syndrome showed normal hemoglobin, but an evidently changed cardiac output and plasma ANP were closely related with the level of the Blood Deficiency. Both parameters might serve as the objective basis to reflect the level of Blood Deficiency to facilitate the clinical diagnosis of the patient.


[Clinical and experimental study on its regulatory function of yi xin decoction (heart-nourishing decoction) to lipids metabolic disturbance in coronary heart disease].

In this research, 74 patients with coronary heart disease (CHD) were grouped in matched-pair, one group took orally Inositol and Mai Tong as the control group, the other group took orally Yi Xin Decoction as the tested group. Indices, i. e. serum levels of apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo-B), high density lipoprotein cholesterol (HDL-c), high density lipoprotein subcomponent cholesterol (HDL2-c), B-lipoprotein (B-LP), total cholesterol (Tch), triglyceride (TG) were measured before and after treatment for 28 days; the results showed that the patients with CHD have prominent derangement of lipid metabolism, which is similar to previous reports. Yi Xin Decoction modified according to Syndrome Differentiation, produced the effect of decreasing the serum Apo-B levels and TG. It also increased Apo-A-1, HDL-c and HDL2-c respectively. Moreover the effect of lowering Apo-B and raising HDL-c in the Yi Xin Decoction group was better than that in the control group. There was no side effect at all; all these indicated that Yi Xin Decoction has a remarkable function of regulating the disturbance of lipid metabolism in CHD patients. In order to further investigate the curative effect of Yi Xin Decoction and elucidate its mechanism, the authors have also investigated Yi Xin Decoction on the experimental mice with hyperlipemia. The result Showed that Tch and TG in atromid and Yi Xin Decoction group reduced after medication, P < 0.01. In comparing with control group, the HDL-c and acidic cholesterol in stool Yi Xin Decoction group rose, P < 0.05. The above study has provided reliable basis for the clinical application of Yi Xin Decoction and also a new medicine to regulate disturbance of lipid metabolism for CHD patients.


[Clinical study of qianxining in the treatment of 60 cases of yang hyperactivity due to yin deficiency type of hypertension].

60 cases with Yang Hyperactivity due to Yin Deficiency type of hypertension were randomly divided into two groups. One was treated with TCM and the other with WM as control. The results showed that: (1) there were no significant differences in the total effective rate and the amplitude of lowering of blood pressure between two groups; (2) the improvement of symptoms and disturbance of autonomic nerve was significant in TCM group in comparison with control; (3) there were some changes in HR, SV, plasma PRA, TXB2 and 6-keto-PGF1 alpha level in both groups, but the decrease of TXB2/6-keto-PGF1 alpha ratio was significant in TCM group only (P < 0.05); (4) TC and TG in patients with hyperlipemia showed a remarkable drop in TCM group (P < 0.02; P < 0.005). All these revealed that Qianxining was a satisfactory hypotensive remedy and a further exploration of its mechanism is suggested.


[Effect of qigong on electrocardiographic autopower spectrum function].

The Changes of positive rate of 17 coronary heart disease cases with frequency domain-correlative cardiogram (FCG) > or = 7 grades were evaluated with electrocardiographic autopower spectrum function before and after Qigong exercise. 17 Qigong-exerciser aged from 54 to 72 (mean 66 year old, male 5, female 12) underwent Qigong exercise in 65 to 103 days and were evaluated using FCG to compare with pre-Qigong exercise status. The results showed that the positive rate of abnormal electrocardiographic autopower spectrum function of lead V5 (Gxx 1/2) decreased from 59% (10/17) to 0% (0/17), the lead II (Gyy 1/2) from 82% (14/17) to 41% (7/17), P < 0.01 and 0.05. This study suggested that Qigong exercise could significantly decrease the positive rate of abnormal electrocardiographic autopower spectrum function and improve perfusion of coronary artery or cardiac dysfunction produced by myocardial ischemia.


[Preliminary study of rose shu-xin oral liquid in the treatment of angina pectoris in coronary heart disease].

200 cases with the Qi Stagnation and Blood Stasis type of coronary heart disease were divided into two groups randomly. Group A used Rose Shu-Xin (heart comforting) oral liquid which is mainly made from the local natural resources-Rose compound products. While group B used Salvia miltiorrhiza (co.) tablet. The results showed that in group A, the total effective rate was 98% and the ECG improving rate was 75%, while in group B, it was 50% and 40% respectively. There was significant difference between group A and B (P < 0.01). Experiments have proved that the Rose oral liquid could improve the myocardial ischemia of the experimental rabbit. It could also reduce the size of infarction area, thus protected the heart from infarction. No adverse action was found in animal experiments and clinical practice. It has proved that the oral liquid could dredge the Liver and regulate the flow of Qi, and remove any obstruction to it. It could also promote the circulation of Blood and relieve pain. It gave the Heart disease a cure from the Liver in TCM theory.


[Experimental study of atherosclerosis and cholelithiasis with the same treatment. I. Effects of yiqi huoxue and shugan liqi agents in atherosclerosis of rabbit].

This paper reports the treatment with Yiqi Huoxue and Shugan Liqi agents in atherosclerosis of rabbit. The results suggested: 1. Both decoctions could reduce the cholesterol of hypercholesterolemia and improve the atherosclerosis, but the former was better than the latter. 2. Both decoctions could alter the components of bile lipids, but on the contrary, the latter was better than the former in reducing the formation of gallstones. 3. Both decoctions could decrease the plasma concentration of LPO and ratio of TXB2/6-K-PGF1 alpha, while increase the ratio of cAMP/cGMP in plasma. So, the different prescriptions of TCM affecting the same link of pathogenesis might play the role of "Different Treatments in Same Disease".


[The effect of Coptis chinensis on lipid peroxidation and antioxidases activity in rats].

In order to make a systematic study of the effect of Coptis chinensis on free radicals, the authors used the method that the drug and the brain homogenate of rat were mixed and incubated to investigate the effect of Coptis on lipid peroxidation. The result showed that the malondialdehyde (MDA) product of rat brain homogenate inhibited by 5% Coptis was significantly different from control (P < 0.001). On the basis of the above-mentioned results, the effect of Coptis on lipid peroxidation and diabetes of rats induced by alloxan was investigated. The result showed: (1) The MDA product of both pancreas and liver homogenate in Coptis group was significantly less than that in control and alloxan group (P < 0.01, P < 0.05). (2) Superoxide dismutases (SODs) in erythrocytes activity was the same for all groups (P > 0.50). (3) The blood catalase (CAT) activity in alloxan group markedly decreased compared with control group (P < 0.05), but no significant change between Coptis and alloxan group (P > 0.05). (4) The value of serum glucose in alloxan group was significantly increased in comparing with control group (P < 0.05). There was a trend to decrease the value of serum glucose in Coptis group compared with alloxan group, but no significant difference between two groups (P > 0.05). The experiment indicated that there was very strong inhibitory effect of Coptis to the lipid peroxidation in vitro and in vivo. Coptis could protect rat from diabetes inducing by alloxan and that probably was due to the fact that Coptis was able to inhibit alloxan inducing free radicals.


[Effect of Salvia miltiorrhiza on the cardial ischemia in rats induced by ligation].

I.p. injection (5 g crude drug/kg) of water extract of Salvia miltiorrhiza to S.D. rats can prevent the acute cardial ischemia induced by the ligation of the coronary artery. The elevation of S-T segment in the electrocardiogram caused by the ischemia was greatly reduced in animals treated with Salvia miltiorrhiza compared with control group. The ischemia area in the left ventricle was significantly reduced and survival rate of the rats increased.


Lasers in periodontics.

Clinical lasers are of two types. Soft lasers are essentially an aid to healing, with relatively few rigorous studies available to support their use. Surgical hard lasers, however, can cut both hard and soft tissues, replacing the scalpel and drill in many areas. After initial experiments with the ruby laser, most clinicians have been using argon, carbon dioxide, and now Nd:YAG systems. The first dental laser based on a Nd:YAG engine provides handpieces of similar size to conventional instrumentation, and being fed by a fibre-optic "cable," has the flexibility for intra-oral use that the carbon dioxide lasers, widely used in oral surgery, lack. Furthermore, extensive clinical investigation has demonstrated their safety in clinical practice, and the fact that procedures can usually be performed without a local anaesthetic is obviously seen as an advantage by patients. Sterilizing as it cuts, the Nd:YAG laser promises to find uses not only in caries removal and soft tissue surgery but also in periodontics and endodontics.


An augmented regenerative technique for severe osseous defects.

Techniques for new attachment using the principle of guided tissue regeneration with barrier membranes have become accepted as a method for treating teeth with severe osseous defects and furcation involvement. After an evaluation of existing membranes and techniques, a procedure was developed using an expanded polytetrafluoroethylene soft tissue patch, 1 mm thick (W. L. Gore and Assoc., Flagstaff, AZ), in conjunction with an alloplast hydroxylapatite bone substitute (HA-500, 40-60 mesh) (Orthomatrix, Minneapolis, MN), to maintain a space for the maturation of the blood clot. An in vitro study of microbial adherence to the soft tissue patch indicated that the number of attached Streptococcus sanguis (gordonii) G9B, and Actinomyces viscosus T14 (V) cells was significantly lower (P < .005 and P < .025, respectively) than the number of bacteria that attached to Gore-Tex Periodontal Material. Two case reports are presented to demonstrate the successful use of the soft tissue patch augmented with hydroxylapatite in patients exhibiting furcation involvement and severe vertical osseous defects.


The fate of collagen during experimental liver lesion with carbon tetrachloride in the presence and absence of colchicine.

Collagen was examined during experimental lesion of the liver with carbon tetrachloride in the presence and absence of colchicine. The experimental animals were Wistar rats, young, of both sexes. The fate of collagen was determined by quantitative assessment of the soluble fraction in salts (NSC), of the soluble fraction in acids (ASC) and of the insoluble fraction (ISC). The results that were obtained indicate protective action of colchicine during the experimental lesion of the liver with CCl4.


Introduction of a neuronal network as a tool for diagnostic analysis and classification based on experimental pathologic data.

A neuronal network, as well as uni- and multivariate statistics and a discriminant analysis were applied to a morphometric database of 58 cases with thyroid neoplasms and normal thyroid tissue. The ability to classify cases correctly according to their diagnosis was compared between the neuronal network and discriminant analysis. For all pairwise comparisons, classification by neuronal network was as least as good as classification by discriminant analysis. For some comparisons, the neuronal network provided more correct diagnoses than discriminant analysis. On the contrary, in a comparison between tumors which are not significantly different according to multivariate statistics, the network reclassifies only half of the cases correctly, whereas discriminant analysis falsely suggests the possibility of classifying cases with either diagnosis. Our results confirm a higher sensitivity of the neuronal network to the diagnostic information contained in the present morphometric database, and we will therefore use this concept for analysis and diagnostic classification in further morphometric studies.


Metastasis induction by incomplete tumor resection. A new metastasis model using inoculation sarcomas in adult nude mice after long-term cultivation of sarcoma cells.

In searching an animal model to study metastasis formation we used cultured cells of experimental rhabdomyosarcomas and their inoculation tumors in adult nude mice. Supplementing earlier observations (Katenkamp et al. 1987) we found that long-term cultured sarcoma cells induce tumors in adult nude mice which do not metastasize spontaneously but produce lung metastases after repeated incomplete tumor removal. Possible factors and mechanisms responsible for metastasis emergence are discussed. The metastasis model introduced may be apt to study cellular changes at cytogenetic and molecular biological level that occur during tumor progression and metastatic dissemination.


Lymphatic absorption of 3-phenylamino-1,2-propanediol and its esters.

From oils associated with the toxic syndrome have been isolated 3-phenylamino-1,2-aminophenol (PAP) and diesters with fatty acids in addition to fatty anilides. These substances have been obtained by chemical synthesis and administered orally to rats whose lymphatic canal had previously been cannulated. The lipid fraction of the lymph collected at different time intervals was isolated. Its chromatographic study has confirmed the presence of the mentioned substances, as well as a metabolite of them, demonstrating that these products enter the blood by lymphatic absorption.


Ultrastructure and cell surface studies of cancer cells following vitamin C administration.

Basal cell carcinomas in rats and squamous cell carcinomas in mice induced by a topical administration of a chemical carcinogen 3-methylcholanthrene (MCA) a single dose of 800 micrograms, were significantly reduced in number and size following a combined administration of vitamin C, a daily dose of 50 mg/kg b.wt. and MCA for 9 months. Ultrastructural studies of basal cancer cells and squamous cancer cells revealed an advanced cytolysis and disorganization of neoplastic cells, mitochondrial alterations, nuclear and nucleolar reduction, and increased phagolysosomes formation, following vitamin C and MCA administration compared to that of neoplastic cells following MCA alone. Scanning electron microscopic observations also revealed cytolysis, increased collagen synthesis, and cell disorganization with membrane disruption following vitamin C and MCA administration, as compared to round basal neoplastic or polyhedral squamous neoplastic cells following MCA treatment alone. These findings demonstrate that vitamin C exerts its antineoplastic effects by increasing cytolytic and autophagic activity, cell membrane disruption, and increased collagen synthesis, and thus, inhibits cancer cell metabolism and proliferation.


Effect of myotonia induced by anthracene-9-carboxylic acid on mitochondrial calcium, plasma creatinine-phosphokinase and aldolase activity in the rat.

The frequent association of myotonia with dystrophy and the knowledge that calcium is increased in injured skeletal muscle cells suggest a possible relationship between cell calcium and myotonic alterations. This investigation has been performed to study the role of calcium in experimental myotonia induced by anthracene-9-carboxylic acid (9-AC) in rats treated with several regimens of food and exercise. Thirty-two rats were divided into 4 groups of 8 rats each, one control and 3 experimental groups. The treatments included caffeine plus exercise (group 2), and a calcium-rich diet (group 3); these procedures were designed to increase intracellular calcium; another group was treated with 9-AC as a myotonia-inducer (group 4). The treatment for all groups lasted 60 days. No significant differences in plasma sodium, potassium, chloride and calcium between control and experimental groups were observed. Whole muscle calcium in wet tissue samples did no change with any treatment. On the contrary, mitochondrial calcium showed a significantly higher concentration in group 3 and 4. CPK and aldolase activities in groups 1, 2 and 3 were similar; but in group 4 these enzyme activities were significantly higher (p less than 0.05). The electrical and mechanical responses were not altered in any rat with any experimental treatment. Our data suggest that myotonia is a predisposing factor for an altered mitochondrial calcium homeostasis in this model; in addition, the enzyme activities of CPK and aldolase were increased in the rats of group 4 implicating that myotonia is a crucial factor in the development of enzymatic abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)


Gross and histologic effects of topical misoprostol on canine gastric mucosa.

This study reports the histological effects of topical misoprostol, a synthetic PGE1 analog, administered in varying dosages on the resting canine gastric mucosa. Misoprostol did not macroscopically or microscopically damage the mucosa but its presumed permeability effects on the gastric vasculature induced marked edema of the mucosa and submucosa. Consistent features included increased thickness of both layers, dilated interglandular regions of the lamina propria, marked subepithelial edema, reduced depth and width of gastric foveolae, vasodilation of the vascular channels, reduced height of surface epithelial cells, swelling of their basolateral intercellular spaces, and increased amounts of surface adherent mucus. It is speculated that the mucosal edema, in addition to an increased mucus layer, may be important in the mechanism of gastric cytoprotection by increasing the distance of penetration or absorption for a mucosal-damaging agent, diluting its concentration, and disseminating any focal accumulations of red blood cells.


Ultrastructural analysis of pulmonary alveolar proteinosis induced by methylnaphthalene in mice.

Pulmonary alveolar proteinosis was induced at a 100% incidence in B6C3F1 female mice by twice weekly painting the back skin with methylnaphthalene for 30 weeks to give a total dose of 7.14 g/kg b.wt. Semithin sections were used for defining areas of type II pneumocyte hyperplasia and hypertrophy and associated proteinosis. Ultrastructurally, alveolar spaces were found to be filled with numerous myelinoid structures resembling the lamellar bodies of type II pneumocytes. Mononucleated giant cells (balloon cells, BC) containing numerous myelinoid structures, lipid droplets and many electron dense amorphous ascicular crystals were closely associated with this extracellular membranous material. Stacks of elastic fibers stained with tannic acid and bundles of collagen fibers were loose and discontinuous in the interstitium of affected areas. The following pathogenesis is hypothesized: type II pneumocytes produce increased amounts of lamellar bodies due to their hyperplasia and hypertrophy and develop to form BC which liberate numerous myelinoid structures on their rupture. Epidermal absorption of methylnaphthalene is an efficient reliable method of induction of this internal disease.


Atypical malignant mesotheliomas with osseous and cartilaginous differentiation after intraperitoneal injection of various types of mineral fibres in rats.

The histopathological appearance of malignant mesotheliomas with osseous and cartilaginous differentiation is described in detail. Bone and cartilage occurred in mixed and sarcomatoid mesotheliomas which were induced by intraperitoneal injection of various types of asbestos fibres (asbestos cement, crocidolite, UICC-amosite, UICC-chrysotile B, Calidria-chrysotile). Bone or cartilage were found in 32.7% of mixed mesothelioma and in 12.8% of sarcomatoid mesotheliomas.


Lipid peroxidation--a common pathogenetic mechanism?

Lipid peroxidation is considered at present as one of the basic mechanisms involved in reversible and irreversible cell and tissue damage. The current knowledge about the role of peroxidative breakdown of polyunsaturated fatty acids in the pathogenesis of various diseases has been reviewed. Lipid peroxidation leads to degradation of the lipid membrane, interaction of degradation products with intra- and extracellular targets and to the production of new reactive oxygen species during the course of the chain reaction thus leading to damage of cells and tissues. According to our current view lipid peroxidation is implicated in the pathogenesis of cancer, inflammatory processes, atherosclerosis, toxic injury by xenobiotics and ischemic-reperfusion damage.


Cardiac metastases of induced lung carcinomas in rats.

A detailed study was made of seven cardiac metastases resulting from induced lung carcinomas. Fifty-six metastasizing lung carcinomas were found in Hann: Wistar rats as a result of subcutaneous injection of DPNA (dipentylnitrosamine). Of these 56, seven (12.5%) metastasized to the heart (three adenocarcinomas and four adenosquamous carcinomas). The metastatic tumors were located in the epicardium, myocardium, or endocardium, and their histologic appearance conformed to that of the primary lung carcinomas. The frequency of the cardiac metastasis and the preference of location within the heart were comparable with human cases.


Effects of N'methyl-N'-nitro-N-nitrosoguanidine and deoxycholic acid on the content of free radicals in rat serum.

The aim of this study was to determine whether changes in serum free radicals may be useful for the early detection of precancerous conditions in the rat colon after treatment with a direct carcinogen (N-Methyl-N'-Nitro-N-Nitrosoguanidine, MNNG) and a secondary bile acid (deoxycholic acid, DCA) as a tumor promoter. It was shown that a significant increase in the concentrations of free radicals in sera of rats following their treatment with MNNG and DCA was observed as early as the 18th week after the beginning of the treatment. Since our results have shown that these alterations occurred in parallel with neoplastic transformations in the rat colon, it suggests that the increase in serum free radicals reflected the precancerous situation in the animals and may be useful in the early detection of cancer development. The possible role of free radicals in deoxycholate-induced liver toxicity was discussed.


Ultrastructural investigations of the enterochromaffin-like (ECL) cells in three different rat strains (Sprague-Dawley, Fischer 344, Wistar) after treatment with the H+,K(+)-ATPase inhibitor pantoprazole.

The purpose of the present study was the evaluation of ultrastructural characteristics of the enterochromaffin-like (ECL) cells in the fundic mucosa of three different rat strains without treatment and after treatment with the H+, K(+)-ATPase inhibitor pantoprazole. In the study, 20 one year old female Sprague Dawley (SD), Fischer 344 (F) and Wistar (W) rats each were treated orally for three months with 4 mg pantoprazole/kg/d or with the vehicle only. The control animals showed close conformity of ECL cell density and morphology in all three strains. Treatment with pantoprazole led to a significant increase in serum gastrin concentration and GPC density in all strains. However, the electron microscopically determined ECL cell density was markedly increased in the SD strain only. Ultrastructurally all treated rats showed activation of the ECL cells, and enhanced histamine release. The SD and F strains had an enhanced proportion of large ECL cell granules, with the F rats also showing an increased granule density. In contrast, the treated W rats were found to have a lower granule density and a higher proportion of small and medium sized granules compared to their controls.


Role of lipid peroxidation in enhancement of endotoxin hepatotoxicity.

The present study was undertaken in the rats to examine whether endotoxin hepatotoxicity is enhanced by increased lipid peroxidation. The rats were given 10 ml of water, corn oil or heated and oxygenated corn oil per kg body weight by stomach tube twice a day for 14 days, and then they were injected physiological saline solution or endotoxin (2 or 2.5 mg per kg body weight) into the tail vein. In the rats pretreated with water or corn oil, the activity of serum glutamic pyruvic transaminase was within the normal limit, and there was no conspicuous morphological change in the liver, except for accumulation of fine fat droplets in few liver cells. On the other hand, in the rats pretreated with heated and oxygenated corn oil, containing a large amount of lipid peroxides, accumulation of small fat droplets in the liver cells and a slight elevation of serum transaminase activity were induced. The challenge with endotoxin (2.5 mg per kg body weight) caused focal hepatocellular coagulative necrosis and a marked elevation of serum transaminase activity, irrespective of the sorts of pretreatment, and there was no significant difference in the biochemical change and the histopathological damage between the rats pretreated with water, corn oil and heated and oxygenated corn oil. These results suggest that increased lipid peroxidation does not contribute to the enhancement of endotoxin hepatotoxicity, although it is thought that carbon tetrachloride and ethanol enhance endotoxin hepatotoxicity by synergism between endotoxin and the chemicals through lipid peroxidation.


Mononuclear cells in normal colon and colonic carcinoma express the same T cell activation markers.

The proportion of mononuclear cells (MC) expressing some of the T cell activation markers in normal colon and colonic carcinomas was determined by immunoperoxidase staining and computer-assisted video image analysis (VIA). Tissue sections were stained with monoclonal antibodies against the T cell activation markers which included the MHC class II antigens DR, DP and DQ, interleukin 2 receptor (CD25) and the p180 (CD45RO) form of the leucocyte common antigen. The mean values for the proportion of leucocytes expressing these activation markers were 92% for DR, 61% for DP, 68% for DQ and 60% for CD45RO in tumours and 90, 62, 70 and 55% in normal tissue respectively. Few cells were stained for the IL2 receptor (mean values 7% for tumours and 5% for normal tissue) or the p220 form (CD45RA) of the leucocyte common antigen (mean values 6% for tumours and 4% for normal tissue). The proportion of MC bearing any of these markers in the normal colon was not significantly different from the matched colonic carcinomas.


Why do women smoke?

This paper will examine the relationship between the dramatic increase of smoking for women while the numbers of male smokers decreases. The powerlessness which characterises women's relationships with all other social groups and individuals is addressed.


Private enterprise occupational health.

A private company in Palmerston North is providing occupational health services to the Manawatu. From an initial concept in mid-1987, the business commenced operations in early 1988. The concept has changed, the shareholding has changed but the business has survived and, by means of plenty of hard work, grew rapidly in 89 and 90, looks likely to achieve a 20% increase in gross turnover in 91/92 and has budgeted for an 11% increase in turnover for 92/93. A.C.C. legislative changes have provided a new opportunity and this is being explored. The new Health and Safety legislation should provide increased opportunities but so far there is little evidence of this occurring. However hope springs eternal and we are confident that we can see through the current hard times and that eventually an improved economy and a settled down health system will allow us to provide increased services for the benefit of the local community.


HIV infection and universal precautions: why are health workers so fearful given the facts?

Identifying people who are infected with HIV has drawn significant media attention over the past decade. This is an ethical issue that must be dealt with by health care workers, urgently. Services to people with HIV are being compromised by the attitudes and practices of some health care workers. Infection control nurses should provide leadership to health care workers who share the challenge of delivering effective and efficient services to people with HIV/AIDS.


A critical reconceptualisation of the environment in nursing: developing a new model.

This paper describes the use of feminist theory and critical social theory in the development of a new nursing model. Basic concepts of the model are defined, and the need for reconceptualisation of the environment is discussed. There is no suggestion that this model is in any way complete; rather it represents an exploration of some of the possibilities. In the second part of this paper the concepts of the model are explored in planning interventions for young women with a smoking habit or with disordered eating.


The detection of oligodeoxynucleotide molecules following uptake into mammalian cells.

A mixed phosphodiester:phosphorothioate oligodeoxynucleotide was used in uptake studies with T15 mouse fibroblast cells. The presence of full-length unlabeled oligomers was identified in both cytoplasmic and nuclear extracts by a method involving gel electrophoresis and electroblotting followed by hybridization with a complementary radiolabeled probe. Detection did not depend on the presence of a label on the oligomer with the potential for its removal, often a problem in other studies. The fate of the oligonucleotides could then be followed with time for at least 3 days. This method of detection should be applicable to studies of nuclease resistance and uptake characteristics of newly developed oligonucleotide analogues.


Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation.

The synthesis of a new class of antisense oligonucleotide compounds with 3'-3' and 5'-5' end inversion (INV-oligonucleotides) is described. Besides the advantage of simplicity of synthesis, physico-chemical studies show that these compounds do not disturb Watson-Crick base-pairing. INV-oligonucleotides have a half-life of 30 h in human serum. We show that they are capable of inhibiting SV40 large T-antigen expression in COS-1 cells, both in vitro and in vivo, and by modulation of the expression of cellular oncoprotein p53 in vitro.


Injection of Antisense RNA specific for E-cadherin demonstrates that E-cadherin facilitates compaction, the first differentiative step of the mammalian embryo.

We have investigated the effect of antisense E-cadherin RNA in the preimplantation mouse embryo. Antisense RNA was injected into each cell of two-cell embryos that were cultured for 2 days until the normal time of compaction and scored for abnormalities. Embryos injected with the antisense RNA showed delayed compaction compared to the embryos injected with control, or sense, RNA. Delayed cleavage was not the cause because nuclear staining with Hoechst dye 33258 showed about the same number of nuclei in both uncompacted embryos injected with antisense RNA compared with compacted embryos injected with sense RNA at the same hours post human chorionic gonadotropin (hCG). Immunofluorescence with a monoclonal antibody to E-cadherin was markedly diminished in embryos injected with antisense RNA compared with control, injected embryos, suggesting that the observed delayed compaction is due to the inhibition of E-cadherin gene expression by antisense RNA. Embryos injected with antisense RNA eventually compacted and then expressed E-cadherin, but at lower apparent levels than controls. Injection of a single blastomere at the two-cell stage created half-embryo abnormalities.


Liposomes as a drug delivery system for antisense oligonucleotides.

Antisense oligonucleotides seem to provide a promising new tool for the therapy of viral diseases and of cancer. However, before the therapeutic potential of antisense compounds can be fulfilled, it will be necessary to overcome significant problems relating to their inefficient uptake by cells and their rapid loss from the body. Phospholipid vesicles (liposomes) have been widely used as a drug delivery system for standard anticancer and anti-infectious drugs. In this article we examine the potential role of liposomes as a drug delivery system for antisense oligonucleotides.


Prognostic implication of gastroparesis in patients with diabetes mellitus.

The prognosis and survival in 13 patients with Type II diabetes mellitus who had delayed gastric emptying as shown by radionuclide tests performed between August 1985 and August 1987 was determined in July 1990. The two patients that were over 80 years of age died within 18 months of the diagnosis of diabetic gastroparesis, but ten of the remaining eleven patients survived. The clinical data on these patients suggest that despite the usual presence of significant co-existent pathology, gastroparesis diabeticorum carries a less ominous prognosis than currently believed.


Cardiovascular reflexes in Parkinson's disease: effect of domperidone and apomorphine.

Cardiovascular reflexes were evaluated in 18 patients with idiopathic Parkinson's disease who had a Hoehn & Yahr score of III-IV. The effect of apomorphine and domperidone on blood pressure, heart rate, R-R interval variation, and the Valsalva ratio were studied. Autonomic dysfunction was not found in the patients and there were no differences between subgroups of patients on different treatments. Apomorphine altered cardiovascular reflexes to a greater degree in patients who received the drug for the first time than in chronically treated patients. The changes were antagonized by domperidone, a peripheral dopamine receptor antagonist. Apomorphine treated patients who were receiving long-term domperidone had similar abnormalities of cardiovascular reflexes to those who had been able to withdraw it.


Cardiovascular responses elicited by simulated diving and their habituation in man.

The cardiovascular responses of 24 subjects were investigated under various simulated diving conditions. Muscle blood flow in forearm and calf, arterial pressure, heart rate and intrathoracic pressure were monitored. Breath holding with face immersion in water at 18 degrees C gave a typical diving response at intrathoracic pressure of 0 and 20 mmHg, (23% bradycardia, greater than 60% muscle vasoconstriction). Breath holding alone at 20 mmHg intrathoracic pressure resulted in vasoconstriction (50%) and bradycardia (4%). Breath holding at 0 mmHg intrathoracic pressure induced a muscle vasoconstriction (5%). These results indicate that both increased intrathoracic pressure and facial immersion can produce a typical diving response individually but that the full 'diving response' requires the presence of both conditions. Diving often activated two responses, the typical 'diving response' and a superimposed defence reaction. Cardiovascular components of the defence reaction (muscle vasodilatation and tachycardia) which was elicited in some divers masked the diving response. In those subjects in whom the diving response was initially absent during repetition of diving manoeuvres the cardiovascular components of the defence reaction were habituated and the characteristic diving response gradually emerged: the initial tachycardia diminished and was replaced by bradycardia, while vasodilatation in the forearm and calf was replaced by vasoconstriction.


Differential effects of isometric exercise on the cutaneous circulation of different regions.

Studies have been made in healthy human subjects of the changes evoked by isometric hand grip in arterial pressure, heart rate and cutaneous red cell flux, the latter being recorded using a laser Doppler meter. Hand grip for 2 min evoked increases in arterial pressure and heart rate, the magnitude of which were graded with the force of contraction (75, 50, or 25% maximum voluntary contraction). Cutaneous red cell flux in the contralateral forearm decreased significantly during 25 and 50% maximum voluntary contraction, while the cutaneous vascular resistance (arterial pressure/cutaneous red cell flux) increased to extents that were graded with the maximum voluntary contraction, indicating graded vasoconstriction. By contrast, cutaneous red cell flux in the face tended to increase, this reaching significance at 75% maximum voluntary contraction. Cutaneous vascular resistance in the face increased in some subjects, but decreased in others, vasodilator responses being most common during 75% maximum voluntary contraction when sweating commonly appeared on the face. In the dorsum of the foot, red cell flux did not change during 75% maximum voluntary contraction, although foot cutaneous vascular resistance increased significantly by the end of the first minute of contraction. At 50 and 25% maximum voluntary contraction most subjects showed an increase in foot cutaneous vascular resistance, but the remainder showed a decrease. We propose that isometric hand grip causes vasoconstriction in the cutaneous circulation of the contralateral forearm, the face and foot, that this response is strongest in the forearm and weakest in the face, and that in the face and foot, the vasoconstriction may be overcome by vasodilatation secondary to sweating.


Reduced vascular excitatory responses to cardiopulmonary unloading in hypertensive patients with left ventricular diastolic dysfunction.

Physiological consequences of altered peak left ventricular diastolic filling rate in hypertension have not yet been fully assessed. The hypothesis that altered left ventricular diastolic filling rate interferes with inhibitory cardiopulmonary reflexes was tested. Normalized peak left ventricular diastolic filling rate was calculated from radionuclide ventriculography. Haemodynamic changes during lower body negative pressure (-5 to -40 mmHg) in nine hypertensive patients with slow normalized left ventricular filling rate (Group A) were compared with 16 hypertensive patients with normal normalized peak left ventricular diastolic filling rate and ten normal volunteers of the same age group. Baseline total peripheral resistance was higher in essential hypertension compared to normals but did not differ significantly between the two hypertensive groups. For data analysis, the levels of lower body negative pressure were grouped as low levels of -5 to -10, and -15 to -20 mmHg, an intermediate level of -25 mmHg, and high levels of -30 to -40 mmHg; the change in total peripheral resistance (from baseline) was less prominent in Group A compared to Group B and to normals (-1.4 +/- 1.7 [SE], -0.06 +/- 1.4, 1.1 +/- 1.2 and 4.5 +/- 2 u.M2 in Group A at the four consecutive levels of lower body negative pressure vs. 0.9 +/- 0.7, 3.8 +/- 0.9, 7.2 +/- 1.6, and 8.2 +/- 1.4 in Group B, and 2.0 +/- 0.7, 3.3 +/- 0.8, 4.9 +/- 0.8, and 5.6 +/- 1.0 in normals). The reductions in central venous pressure and in pulmonary wedge pressure were not significantly different among the three groups at the different levels of lower body negative pressure, but the reduction in cardiac output was smaller in patients with reduced dv/dt ratio than in the other two groups. The responses to the cold pressor test were similar in all subjects. We conclude that patients with essential hypertension and diastolic dysfunction have impaired total peripheral resistance responses to lower body negative pressure. This abnormality may reflect an alteration in cardiac baroreflexes secondary to left ventricular diastolic dysfunction, an influence of baseline sympathetic activity on the observed vascular responsiveness to lower body negative pressure, or primary differences among groups in the changes in cardiac output induced by similar levels of lower body negative pressure.


The long-term reproducibility of clinical tests of autonomic cardiovascular function in normal man.

Tests for the integrity of autonomic cardiovascular reflexes have been widely used in the clinic and in space physiology for decades. However, whereas some information on the short-term reproducibility of such tests are available, little is known about their long-term reproducibility. The work in this study was, therefore, directed towards assessing intra- and inter-subject variations in responses (heart rate, mean arterial blood pressure, forearm blood flow and forearm vascular conductance) to cortical arousal, cold face stimulation and lower body negative pressure (at 10, 30 and 50 mmHg) in eleven healthy male subjects (aged between 22 and 45 years). Subjects were studied repeatedly (each month) over a 6-month period. It was found that forearm vascular conductance responses to cold face stimulation were the most reproducible (mean coefficient of variation 10.9%), and with diminishing reproducibility curve responses to lower body negative pressure at 50 mmHg (mean coefficient of variation 12.4%), lower body negative pressure at 30 mmHg (mean coefficient of variation 18.9%), lower body negative pressure at 10 mmHg (mean coefficient of variation 28.0%), and responses to cortical arousal (mean coefficient of variation 39.6%). Generally, subjects who showed the largest responses to cold face stimulation also showed the largest responses to the other tests, and vice versa. It is concluded that there is intra-individual variability in the responsiveness and reproducibility of cardiovascular tests and that the cardiovascular responses to cold face stimulation and lower body negative pressure at 50 mmHg are the most reproducible.


The effect of time of day on orthostatic tolerance and the cardiovascular effects of a high carbohydrate meal in healthy young subjects.

Ingestion of a high carbohydrate meal leads to a fall in blood pressure but does not change orthostatic tolerance in healthy elderly subjects. Much smaller effects are observed in young subjects, but studies have only been performed in the morning. The purpose of this study was to compare orthostatic responses and the effect of a high carbohydrate meal in the morning and in the afternoon in young subjects. Fourteen healthy, young subjects (four female, mean BMI = 22.0 kg/m2, age range 21-27 years) were studied on two occasions, in the morning (0830 h) and in the afternoon (16.30 h) on separate days. Blood pressure, heart rate and cardiac output were measured noninvasively before and after the ingestion of a standard meal with 76-77% of the energy being provided by carbohydrate. Time of day had no effect on blood pressure and heart rate responses to tilting after food. Cardiac output fell significantly during tilting in the fasted state. In the morning there was a progressive fall in cardiac output with a decrease of 0.83 l/min at 45 degrees (95% confidence interval of the change -1.37 to -0.27 to l/min) and a fall of a further 0.15 l/min at 75 degrees. By contrast in the afternoon cardiac output fell 0.84 l/min at 45 degrees (95% confidence interval of the change -1.31 to -0.35 l/min) and then rose by 0.61 l/min on tilting to 75 degrees (95% confidence interval of the change +0.22 to +0.98 l/min. Interaction effect p = 0.02 ANOVA). Supine cardiac output increased after food ingestion at both times of day (p less than 0.01 ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)


Parasympathetic denervation of the iris in Chagas' disease.

The parasympathetic innervation of the iris along with cardiovascular reflexes involving parasympathetic and sympathetic function were studied in 45 patients with Chagas's disease and in 36 controls. The autonomic features in Chagas' disease included 15 with cardiomyopathy, three with megaoesophagus and five with megacolon. None of the patients had orthostatic hypotension. Parasympathetic cardiac reflexes (deep breathing 30:15 ratio, Valsalva) were abnormal. There were exaggerated pupillary responses to dilute pilocarpine. These studies suggest iris parasympathetic denervation and favour more widespread cholinergic involvement in patients with Chagas' disease, than previously recognized.


Severe paroxysmal hypertension induced by a subconscious Valsalva-like manoeuvres.

We describe the case history of a 32 year old male with severe paroxysmal hypertensive spikes when there was an increase in diastolic blood pressure to 150-200 mmHg. These spikes occurred during rest and especially during modest exercise. They were associated with headache and dyspnoea and were resistant to antihypertensive medication. After 5 years of observations and investigations he underwent further 24-h intra-arterial blood pressure monitoring and physiological testing. The 24-h blood pressure profile was near normal at rest. The observed hypertensive spikes seemed to be induced by involuntary Valsalva-like manoeuvres. This had not been detected previously by the investigators and the patient was unaware of inducing these manoeuvres.


Alcohol and drug abuse in Nepal.

Alcohol use has been in Nepal since time immemorial. Social tolerance to alcohol use is quite high and so far alcohol has not been taken seriously either by the Government or by any social organization. Production, sale, and consumption of alcohol is ever on the increase and it could be taken as the number one problem drug in the country. Cannabis and opium use has been in Nepal for centuries and in the past they did not pose much of a problem. Drug use began to be seen as a problem since only the mid-1960s and early-1970s with the influx of large numbers of hippies. Presently, the drug scene in Nepal is dominated by heroin and it has affected youths, mainly in the urban areas. A number of measures, both on supply reduction and demand reduction, have been taken by the Government together with non-Governmental organizations. However, the number of drug users is on the increase. Relapse rate following detoxification treatment is quite high. After-care and rehabilitative measures are lacking. Many drug users become involved in high-risk behaviours in spite of their knowledge of the dangers. Therefore, it calls for the change in our strategies which must be based on the thorough understanding of human nature and its behaviour.


Implications of gender for alcohol treatment research: a quantitative and qualitative review.

Previous reviews of alcohol treatment research have indicated that in the majority of studies there are no sex differences in treatment outcome. The current meta-analysis was used to measure the magnitude and direction of trends of sex difference in treatment outcome. The results indicated that women had better treatment outcomes than men in the first 12 months after treatment while men showed greater improvement than women in follow-ups after 12 months. However, the estimated differences were small and derived from a heterogeneous sample of studies. Evidence from the studies in the meta-analysis is used to highlight the importance of gender-related factors which may impact on the processes and outcomes of treatment. In particular, sex differences in physiological responses to alcohol, in social norms for alcohol, and in socio-cultural experiences are considered important areas for future investigation in alcohol treatment research.


User reports of problems associated with alcohol and marijuana.

A general population sample of 5126 New Zealanders aged 15-45 in two regions was surveyed to determine their use of alcohol, tobacco, marijuana, and other illicit drugs. Self-reported problems were recorded on identical scales for alcohol and marijuana in order to make a direct comparison between alcohol and marijuana-related problems. The results suggest that alcohol-related problems were more common than marijuana-related ones in the general population, reflecting the fact that alcohol use was more widespread. Within the 17% of the sample who had used both alcohol and marijuana in the last 12 months more problems were reported from alcohol, once again reflecting differences in amounts consumed. Among heavier users of marijuana both alcohol and marijuana-related problems were more commonly reported than in the general sample. Problems were reported at similar levels for both drugs and the pattern of problems is somewhat similar.


Predicting initiation to and cessation of buprenorphine and temazepam use amongst adolescents.

Two interviews separated by 12 months (mean) were conducted with a purposive sample of Glaswegian adolescent drug misusers. Across that interval, beginning to use buprenorphine and temazepam were predictable by prior extent of other substance use, especially cannabis and tobacco, and by the Drug Misuse Scale, using selected MMPI items. About 50% of those who had been using either drug prior to first interview did not use at all between first and second interview. For temazepam, this cessation was not predictable, but ceasing to use buprenorphine was more likely the less time subjects had used this drug and if they had never injected. It is concluded that personality and frequent substance use predispose to drug misuse, but that additional factors must lead to dependence.


Cigarette smoking and cognitive performance.

While some investigations into the relationship between smoking and cognitive performance have reported that smoking facilitates performance, other research has come to the opposite conclusion. A review of the literature suggests that this variance in results may be due to differences among studies in design (comparing smokers only with deprived smokers rather than with non-smokers) and also to differences in task demands. Therefore, performance of smokers having just smoked, matched smokers deprived for a brief period, and also non-smokers was contrasted on a series of tasks which ranged from repetitive and perceptually-bound tasks to complex, dynamic tasks dependent upon long-term memory. It was found that while cigarette smoking had no negative effect upon performance for simple perceptual tasks, smoking was found to exert measurable negative effects upon performance for more complex information processing tasks.


The anatomy of a follow-up.

This article describes the methods that were used to maximize subject retention in an 8-12 year follow-up study of 454 men. Guidelines and suggestions are given regarding follow-up structure, strategies to relocate lost subjects, and efforts to maintain contact and ensure subject cooperation. Through these methods all 454 subjects have been located, and at the present time an estimated 98% or greater will be completely evaluated at follow-up. Hopefully these suggestions will be helpful to researchers and clinicians interested in longitudinal studies.


Predictors of smoking behaviour: an application of Ajzen's theory of planned behaviour.

The aim of the present paper was to verify the basic assumptions underlying the theory of planned behaviour for the prediction of cigarette smoking intentions and behaviour among adults of the general population (study 1) and a group of pregnant women (study 2). Each study was developed based upon Ajzen's theory of planned behaviour. In both studies, baseline data was collected at home with trained interviewers and with the use of paper and pencil questionnaires. The self-report on behaviour was obtained 6 months (study 1) and between 8 and 9 months (study 2) after baseline data collection. In study 1, for smokers, perceived behavioural control, attitudes and subjective norm were explaining intention, whereas perceived behavioural control and habit were the most important predictors of behaviour. In study 2, smoker's intentions was mainly under the influence of perceived behavioural control and attitude, whereas behaviour was predicted by perceived behavioural control only. The present studies suggest that promotional programmes should help smokers to know and develop their will-power regarding non-smoking of cigarettes and should be informed of the effort required in order to modify smoking behaviour.


The incidence of illicit drug use in the United States, 1962-1989.

Epidemiological descriptions of drug abuse in the US in the last three decades have generally not included data on the patterns and trends in the incidence of illicit drug use (i.e. new users). In this paper, estimates of illicit drug use incidence are presented, based on retrospective data from the National Household Survey on Drug Abuse. Incidence of marijuana use began increasing in the 1960s and reached a peak in 1973, after which a continuing decline was seen. Cocaine use incidence began to increase in the late 1960s and reached a peak in 1982, then declined.


Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities.

ELISA assays have been developed for alpha(1-3)N-acetylgalactosaminyltransferase (blood group A transferase) and alpha(1-3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc alpha 1-2Gal beta-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc alpha 1-2(GalNAc alpha 1-3)Gal beta-R-BSA or Fuc alpha 1-2(Gal alpha 1-3)Gal beta-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.


Detection of the ganglioside N-glycolyl-neuraminyl-lactosyl-ceramide by biotinylated Escherichia coli K99 lectin.

K99 lectin from Escherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognize N-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay.


Production and characterization of a monoclonal antibody (BBH5) directed to ganglioside lactone.

The occurrence of lactones in various ganglioside preparations has been clearly demonstrated, yet the natural occurrence of ganglioside lactones in cells and tissues has been the subject of long debate, since lactones can be formed readily during preparation of gangliosides. We now report the generation of monoclonal antibody (BBH5) that reacts specifically with lactones of disialogangliosides having the NeuAc2-8NeuAc2-3Gal sequence, but does not crossreact with the parent ganglioside. The specificity of the antibody resides on the first lactone ring between two sialic acid residues but not on the second lactone ring between sialic acid and galactose, as evidenced by reactivity with lactonized GD1b having the first lactone ring (L1), and by reactivity with lactonized polysialic acid homo-oligomers ([NeuAc alpha 2-8]nNeuAc). The sialic acid carboxyl involved in the lactone ring was unequivocally determined after ammonolysis followed by methylation and fast atom bombardment mass spectrometry. The antibody BBH5 thus provides a novel tool for studies of the natural occurrence of lactones in cells and tissues.


Expression, glycosylation and secretion of yeast acid phosphatase in hamster BHK cells.

The gene PHO5 coding for one of the repressible acid phosphatases of the yeast Saccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human beta-actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular mass M(r) = 62,000, indicating substitution of the polypeptide moiety by 2-3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.


Sequence analysis of p-hydroxyphenyl-O-beta-D-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts.

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated with p-hydroxyphenyl-beta-D-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After 125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment delta HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments delta HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on the N-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-A, Havsmark B, Silverberg I (1990) Biochem J 269:381-8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.


Application of 2D and 3D NMR experiments to the conformational study of a diantennary oligosaccharide.

By the application of homonuclear 3D NOE-HOHAHA and heteronuclear 3D HMQC-NOE experiments in studies of complex oligosaccharides, NOEs can be investigated which are hidden in conventional 2D NOE spectra. In the 3D NOE-HOHAHA spectrum omega 3 cross sections were considered to be the most suitable for assignment of NOEs. Alternatively, these cross sections could be measured separately in selective 2D HOHAHA-NOE spectroscopy. The advantages and limitations of the 2D alternative are compared with those of the 3D NOE-HOHAHA approach. In 3D HMQC-NOE spectroscopy the larger chemical shift displacement of the carbon spectrum with respect to the proton spectrum can be used to unmask NOEs hidden in the bulk region. If the extra proton dimension is not needed, 2D HMQC-NOE is a good alternative. The suitability of 2D and 3D NOE-HOHAHA and HMQC-NOE experiments for the estimation of proton-proton distances is demonstrated by comparing the results of these experiments on a diantennary asparagine-linked oligosaccharide with those of a conventional 2D NOE experiment. NOEs identified in the 2D and 3D NOE-HOHAHA as well as HMQC-NOE experiments, so far not identified or not quantified in 2D NOE experiments, are discussed in relation to each glycosidic linkage. The flexibility of the Man alpha (1-3)Man linkage is demonstrated, confirming the existence of an ensemble of conformations for this linkage.


Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy.

Non-glycine residues with positive theta-angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant (3)JHNHalpha and integration of the nuclear Overhauser HN-Halpha cross peak, positive theta-angles could be determined reliably to 60 degrees +/- 30 degrees, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive theta-angles can also occur in non-glycine residues and in the four proteins, positive theta-angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured (3)JHNHalpha coupling constants and the intensity of the intraresidue HN-Halpha NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wuthrich, K. (1984) J. Mol. Biol., 180, 741-751; Ludvigsen, S. Andersen, K.V. and Poulsen, F.M. (1991) J Mol. Biol., 217, 731-736).


Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.


Precise vicinal coupling constants 3JHN alpha in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments.

Improved experimental schemes for the recently introduced J-modulated [15N,1H]-correlation experiment for measurements of the homonuclear amide proton-C alpha proton vicinal coupling constants, 3JHN alpha, in uniformly 15N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of 3JHN alpha. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M = 7.3 kDa), where at 13 degrees C precise values of 3JHN alpha in the range 2.0-9.5 Hz were obtained for all residues with resolved 15N-1H cross peaks. It was then applied to the Antennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex approximately 18 kDa). The 3JHN alpha values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.


Epidemiology of pediatric brain tumors.

Brain tumor research is unlike some other areas of cancer research, in which definite causes have been identified and prevention and treatment strategies may be developed. Primary brain tumors are heterogeneous with respect to several characteristics, and brain tumor research is hampered by this fact, as well as by the small numbers of cases, by the lack of a clearly established and consistently applied histopathologic classification scheme, and by geographical variations in diagnostic capabilities and mechanisms for case reporting. The current movement toward molecular epidemiology has resulted in several changes in the ways we will be able to investigate cancer etiologies in the future, and promises to be especially fruitful in the area of brain tumor research. The search is under way for biologic markers of risk exposures for various cancers, and for laboratory methods to identify those individuals who, because of their genetic qualities, are most likely to have brain tumors. There are probably multiple causes for brain tumors, incorporating both genetic and environmental pathways. Prior research has consistently identified few risk factors for brain tumors, such as ionizing radiation. Thus, collaborative efforts among clinicians, laboratory scientists, and epidemiologists, which make use of molecular epidemiologic methods, are perhaps of greater importance in this field than for cancers arising at other sites.


Structure and conformation in solution of the parallel-stranded hybrid alpha-d(CGCAATTCGC).beta-d(GCGTTAAGCG) by high-resolution 2D NMR.

The unnatural duplex oligonucleotide alpha-d(CGCAATTCGC).beta-d(GCGTTAAGCG) was analyzed by high-resolution NMR methods. All of the exchangeable imino and nonexchangeable protons of the duplex were assigned. Detection of all 10 of the exchangeable imino protons confirms that a parallel, unsymmetrical duplex is formed. The thermal stability of the parallel duplex is similar to the analogous antiparallel beta-beta duplex. The right handedness of the helix is confirmed by inter-residue [H8/H6-H1'] and [H8/H6-H2"] NOEs to the 5'-neighbor in the beta-strand and [H8/H6-H1'] NOEs to the 3'-neighbor in the alpha-strand. Intra-residue and inter-residue distances between base protons and deoxyribose protons in both strands were determined using the isolated spin-pair approximation for NOESY cross peaks acquired with mixing times 50 ms or less. The NOE data are consistent with a B-form geometry adopted by the alpha/beta hybrid decamer.


Neuroimaging of pediatric brain tumors.

This article covers techniques of imaging, posterior fossa tumors, and supratentorial tumors. Computed tomography and magnetic resonance imaging are commonly used in the diagnosis and evaluation of pediatric brain tumors. MR imaging is the primary imaging technique because it is more sensitive in the detection of small tumors, particularly those in the posterior fossa.


Neurofibromatosis and central nervous system tumors in childhood.

Neurofibromatosis is a multifaceted disease that often results in tumors of the central nervous system. As our understanding of the molecular biology of the disease improves along with better neuroradiology imaging, surgical instrumentation, and adjunctive care, the management schemes for these patients are evolving. This article reviews what is known about neurofibromatosis, common management problems with respect to the central nervous system, and an approach to the handling of these issues.


Infant brain tumors.

This article describes the histology and location of brain tumors in young children as well as the presenting features of tumors in this age group and then focuses on three tumor types: medulloblastoma, ependymoma, and chiasmatic optic glioma. The discussion proceeds in terms of prognosis, late effects of treatment, and current and future strategies for treatment.


Suprasellar and sellar tumors in childhood and adolescence.

Suprasellar and sellar region tumors in children constitute a diverse group of lesions. We advocate an aggressive surgical approach to these tumors for diagnostic and therapeutic purposes. With detailed knowledge of the microneurosurgical anatomy of this region, the neurosurgeon can choose from a number of operative approaches on the basis of tumor size and location. The benefits of radiation therapy for craniopharyngiomas and optic chiasmatic/hypothalamic tumors must be carefully weighted against the substantial long-term risks associated with irradiation of the developing brain in this region. During the next decade, the role of primary chemotherapy for germinoma and adjuvant chemotherapy for optic pathway gliomas will be determined. Also, the role of stereotactic radiation therapy, of great value to some children with recalcitrant tumors in the suprasellar region, may be better defined.


Midline supratentorial tumors.

Tumors of the supratentorial midline are common in children and are often amenable to an aggressive operative resection. MR imaging is invaluable to planning an operative strategy. A variety of surgical corridors provide access to the deep midline structures, notably the transcallosal, transventricular, and basal routes. The histology is important in determining the value of adjuvant treatments, such as radiation therapy and chemotherapy. These complex tumors are best managed by a multidisciplinary group with a special interest in pediatric neuro-oncology.


Pineal tumors.

Tissue diagnosis is necessary for optimal treatment of pineal region tumors in children. Preoperative staging should include craniospinal MR imaging with and without gadolinium DTPA enhancement, CSF sampling for cytology, and measurement of biologic tumor markers in serum and CSF. Surgical approach is determined by results of preoperative MR imaging and the extent of resection by the results of staging and intraoperative frozen-section histopathologic evaluation. There is no longer a role for the radiation test dose (2000 cGy) in the management of these tumors. Postoperative treatment is based on histopathology and extent of disease. Benign tumors are treated with surgery only, and nondisseminating focal tumors with surgery and focal radiation therapy. Non-germinoma malignant germ cell tumors are best treated with neoadjuvant chemotherapy followed by radiation therapy given focally for focal disease; craniospinal radiation therapy is reserved for patients with evidence of disseminated disease at the completion of induction chemotherapy.


Brainstem gliomas.

Brainstem gliomas, a relatively common form of childhood brain tumor, are highly resistant to therapy. With computed tomography and magnetic resonance imaging, these lesions can be diagnosed with a high degree of reliability. The indications for surgery are unclear. Focal lesions may be amenable to partial resections. Stereotactic approaches can be used for diffuse lesions, but it has not been shown that the information obtained changes the approach to treatment or outcome. Higher dose radiotherapy has been recently used but has not improved survival for most patients. Patients with brainstem gliomas must be stratified into risk groups, and new means of treatment are needed.


Posterior fossa tumors.

Cerebellar and fourth ventricle tumors are usually associated with hydrocephalus, which is now treated with ventriculostomy and tumor removal so that permanent shunts are needed in only one third of children. Complete removals decrease the recurrence rate of cerebellar astrocytomas and ependymomas. Postoperative staging with spinal magnetic resonance imaging has decreased the need for myelography. Adjuvant chemotherapy improves the survival of children with high-stage medulloblastomas.


Pediatric skull, skull base, and meningeal tumors.

Calvarial neoplastic and non-neoplastic tumors are routinely encountered by all neurosurgeons. Benign and malignant skull base and meningeal tumors are relatively rare lesions in children. Interdisciplinary approaches to those tumors more frequently encountered in the pediatric population in these locations are discussed. Unique aspects of the diagnosis, treatment, and prognosis for infants and children are discussed.


Spinal cord compression by epidural tumors in childhood.

Epidural malignancy is the most common cause of nontraumatic spinal cord compression in children. Many different tumors may cause compression, either at presentation or sometime during treatment. Treatment options include surgery, irradiation, and chemotherapy. The decision as to the correct option depends on the tumor type and the degree of compression present.


Intramedullary spinal cord tumors in children.

Intramedullary spinal cord tumors are uncommon in children, and delayed diagnosis is common. Unlike the situation in adults, low-grade astrocytomas predominate followed by ependymomas and other gliomas. Recent technological advances, including MR imaging, intraoperative ultrasonography, the ultrasonic surgical aspirator, and the operative laser, have made radical resection of intramedullary tumors feasible. The long-term results of modern-day surgery using these techniques are only now becoming available, and the role of adjuvant radiotherapy or chemotherapy for the usual low-grade tumors remains unclear. The risk of spinal deformity after laminectomy in children and the effects of radiotherapy on the growing spine create special challenges in the treatment of pediatric intramedullary tumors.


Pediatric spinal axis tumors.

Pediatric spinal cord tumors occur in the intramedullary or extramedullary spaces. The extramedullary tumors are further divided into those in intradural-extramedullary or extradural locations. Tumors in the intradural-extramedullary region include nerve sheath tumors, meningiomas, and "embryonal" tumors. In the extradural space are neuroblastomas, sarcomas, and other primary tumors of bone. The radiographic findings, histology, and management of each type of tumor are included in this article, which focuses on extramedullary tumors.


Long-term effects of treatment for childhood brain tumors.

Recent studies have recognized brain tumor to be the most important solid malignancy in childhood. Improved diagnostic methods and advances in surgical technology and instrumentation have significantly lowered operative mortality, facilitating more precise and radical surgical resections.


Cutinase is not required for fungal pathogenicity on pea.

Cutinase, a fungal extracellular esterase, has been proposed to be crucial in the early events of plant infection by many pathogenic fungi. To test the long-standing hypothesis that cutinase of Nectria haematococca (Fusarium solani f sp pisi) is essential to pathogenicity, we constructed cutinase-deficient mutants by transformation-mediated gene disruption of the single cutinase gene of a highly virulent N. haematococca strain. Four independent mutants were obtained lacking a functional cutinase gene, as confirmed by gel blot analyses and enzyme assays. Bioassays of the cutinase-deficient strains showed no difference in pathogenicity and virulence on pea compared to the wild type and a control transformant. We conclude that the cutinase of N. haematococca is not essential for the infection of pea.


Acquired resistance in Arabidopsis.

Acquired resistance is an important component of the complex disease resistance mechanism in plants, which can result from either pathogen infection or treatment with synthetic, resistance-inducing compounds. In this study, Arabidopsis, a tractable genetic system, is shown to develop resistance to a bacterial and a fungal pathogen following 2,6-dichloroisonicotinic acid (INA) treatment. Three proteins that accumulated to high levels in the apoplast in response to INA treatment were purified and characterized. Expression of the genes corresponding to these proteins was induced by INA, pathogen infection, and salicylic acid, the latter being a putative endogenous signal for acquired resistance. Arabidopsis should serve as a genetic model for studies of this type of immune response in plants.


Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.


Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein.

By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.


Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.


A single amino acid substitution in the coat protein of cucumber mosaic virus induces chlorosis in tobacco.

Some strains of cucumber mosaic virus (CMV) induce a bright yellow/white chlorosis in tobacco instead of the light green/dark green mosaic induced by most CMV strains. This property is controlled by RNA 3 of this tripartite virus. Recombination between cDNA clones of RNA 3 from a green mosaic strain, Fny-CMV, and a chlorotic strain, M-CMV, and inoculation of infectious transcripts of the chimeric RNAs 3, together with RNAs 1 and 2 of Fny-CMV, localized the chlorosis induction domain to a region of the coat protein gene containing two nucleotide differences. Site-directed mutagenesis of one nucleotide to change the codon for Leu129 in the M-CMV coat protein to Pro129 of Fny-CMV changed the phenotype from chlorotic to green mosaic, whereas the opposite change in phenotype was observed when the Pro129 in the Fny-CMV coat protein was altered to Ser129. Thus, the local secondary structure surrounding amino acid 129 rather than a particular amino acid per se is involved in chlorosis induction.


Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.


A novel light-regulated promoter is conserved in cereal and dicot chloroplasts.

The chloroplast psbD-psbC genes encode D2 and cp43, a reaction center protein and chlorophyll-binding antenna protein of photosystem II, respectively. We have previously shown that differential accumulation of light-induced psbD-psbC mRNAs in barley chloroplasts is due to transcription from a blue light-responsive promoter (LRP). It is hypothesized that the light-induced mRNAs help to maintain levels of the D2 polypeptide, which is photodamaged and degraded in illuminated plants. To determine if light-induced accumulation of psbD-psbC mRNAs was a conserved phenomenon in chloroplasts, the expression of psbD-psbC operons from five cereals (barley, wheat, rice, maize, and sorghum) and three dicot (tobacco, spinach, and pea) species was examined. Cereal and dicot psbD-psbC operons differ due to several DNA rearrangements that moved psbK-psbI proximal to psbD-psbC, allowing cotranscription of these genes and production of several unique transcripts in cereals. Despite differences in the structure and expression of the cereal and dicot psbD-psbC operons, the accumulation of light-induced psbD-psbC mRNAs was conserved in all species studied. An unusual feature of the light-induced mRNAs was the occurrence of 5' end microheterogeneity. The multiple 5' termini were mapped to several consecutive nucleotides (8 to 25 bp) within a highly conserved (61%) DNA region that represents the transcription initiation site for the mRNAs in barley and tobacco. The novel LRP differs in sequence from typical plastid promoters that have prokaryotic "-10" and "-35" elements and is centered 570 bp (cereals), 900 bp (tobacco, spinach), or 1100 bp (pea) upstream from the psbD translational start codon. We propose that physiological and gene regulatory demands of the chloroplast act as constraints that preserved the linkage of the LRP with psbD despite DNA inversions involving the psbD upstream region.


Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.

The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and squash leaf curl virus (SqLCV) have two single-stranded circular genomic DNAs, the A and B components, thought to be replicated from double-stranded circular DNA intermediates. Although it has been presumed that the origin sequences for viral replication are located in the highly conserved 200-nucleotide common region (CR) present in both genomic components and that the viral-encoded AL1 protein interacts with these sequences to effect replication, there has been no evidence that this is in fact so. We have investigated these questions, demonstrating selectivity and sequence specificity in this protein-DNA interaction. Simple component switching between the DNAs of TGMV and SqLCV and analysis of replication in leaf discs showed that whereas the A components of both TGMV and SqLCV promote their own replication and that of their cognate B component, neither replicates the noncognate B component. Furthermore, using an in vivo functional replication assay, we found that cloned viral CR sequences function as a replication origin and direct the replication of nonviral sequences in the presence of AL1, with both circular single-stranded and double-stranded DNA being synthesized. Finally, by the creation of chimeric viral CRs and specific subfragments of the viral CR, we demonstrated sequence-specific recognition of the replication origin by the AL1 protein, thereby localizing the origin to an approximately 90-nucleotide segment in the AL1 proximal side of the CR that includes the conserved geminiviral stem-loop structure and approximately 60 nucleotides of 5' upstream sequence. By deletional analysis, we further demonstrated that the conserved stem-loop structure is essential for replication. These studies identify the functional viral origin of replication within the CR, demonstrating that sequence-specific recognition of this origin by the AL1 protein is required for replication.


A tobacco DNA binding protein that interacts with a light-responsive box II element.

Ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in photosynthetic carbon fixation in higher plants. The small subunit of this chloroplast enzyme (rbcS), encoded by a family of nuclear genes, is regulated at the transcriptional level by light. Promoter analyses have previously identified the box II sequence as a cis element critical for the light-regulated expression of rbcS genes. Nuclear factor GT-1 binds specifically to this element and is one of the plant nuclear factors that has been detected and studied in great detail. Here we describe the cloning and characterization of a tobacco cDNA encoding a protein, designated B2F (Box II Factor), with similar binding specificity and mobility in gel retardation assays as nuclear GT-1. Steady state levels of mRNA encoding B2F do not appear to be regulated by light; this is consistent with the previous observation that nuclear GT-1 activity is present in extracts from both light-grown and dark-adapted plants. Sequence comparison with another plant trans-acting factor, GT-2, which binds to a GT-like element in the rice phytochrome promoter, shows striking homology in three putative alpha-helices that may be involved in DNA binding.