Membranous nephropathy: a 19 year prospective study in 51 patients.

to study prospectively patients presenting with membranous nephropathy. the outcome of 51 patients (33 male; mean age 50.8 SD 17.7 years; 49 caucasian, two Maori), aged 14 years and over, with membranous nephropathy was studied prospectively. The patients were enrolled between 1 July 1972 and 30 June 1991. Patients with systemic lupus erythematosus were excluded. Membranous nephropathy was secondary to drug therapy in eight (gold 3, nonsteroidal antiinflammatory drug 3, penicillamine 1, captopril 1). All patients were hepatitis B surface antigen negative. The majority of patients did not receive immunosuppressive therapy. forty-seven (92%) presented with the nephrotic syndrome, eight (16%) with a plasma creatinine > or = 0.15 mmol/L and 28 (55%) with hypertension. The patients were followed for a mean of 5.4 years, the median being 3.9 years and range four months-19 years. The 10 year cumulative renal survival was estimated to be 78% and overall survival 66%. Six patients have entered a renal replacement programme and six have died of nonrenal causes. Of the remaining 39, eight have renal insufficiency, 27 hypertension and 22 proteinuria. Complete remission occurred in 17 (33%)-11 spontaneously, four following withdrawal of the offending drug and two after immunosuppressive therapy. Poor prognostic indicators at presentation included renal insufficiency, need for antihypertensive therapy, pathological stage IV on renal biopsy and older age. membranous nephropathy is an indolent disease with a good chance of spontaneous remission. In most patients immunosuppressive treatment appears unwarranted.


Attitudes to specialist recertification: results of a national survey among pathologists.

to evaluate attitudes and preferred mechanisms in the establishment of specialist recertification for pathologists. a national survey was conducted amongst the membership of the New Zealand Society of Pathologists. responses were received from 54 (62%) financial members of the society. The majority of respondents agreed with the concept of compulsory periodic recertification. The ideal recertification period should be 10 years and the system should be supervised by the New Zealand Society of Pathologists. The favoured recertification mechanism was by continuing medical education which should be funded by the individual candidate. The majority of respondents supported the establishment of voluntary recertification on a trial basis. the survey suggested a favourable attribute amongst pathologists to the introduction of continuing education based periodic recertification.


Health screening in a general practice by opportunistic recruitment.

this study aims to assess the effectiveness of opportunistic recruitment into a general practice health screening programme and subsequent improvement in screening rates. a study group of 754 patients aged 30-69 years (345 men, 409 women) in a Wanganui general practice were invited by the doctor when attending the surgery to return for a preventive health check. The check included a self administered screening questionnaire, selected examination items performed by the practice nurse, and a consultation with the doctor to review the findings. over the three year study period 327 of the men and 392 of the women attended the surgery. An invitation to return for a health check was offered to 307 men and 329 women with completion of health checks by 294 men and 317 women, resulting in a screening rate in the study group of 85% for men and 78% for women. Selected screening indicators showed improvement with blood pressure recording increasing from 48% to 85% for men, and cervical cancer screening improving from 45% to 87% for women. opportunistic recruitment by the doctor's personal invitation proved an effective approach to health screening in this practice with significant improvement in selected screening indicators.


Potential applications of high-dose megestrol acetate in breast cancer.

Empiric clinical trials have revealed new mechanisms by which hormonal therapies may exert their antitumor effects. Initial studies using escalated doses of agents like toremifene and megestrol acetate have yielded interesting results, showing responses in hormone-receptor-negative patients and in patients progressing after standard doses, respectively. A trial by Cancer and Leukemia Group B randomizing patients with advanced breast cancer to standard-dose (160 mg) megestrol acetate or to 5 or 10 times the standard dose (800 and 1,600 mg) has completed accrual. It is hoped that these results will provide a definitive answer to the dose-response issue for breast cancer. However, regardless of this trial's ultimate outcome, higher doses of megestrol acetate have demonstrated important new effects on appetite stimulation and weight gain; ongoing laboratory research promises potential roles for megestrol acetate in the reversal of chemotherapeutic drug-induced tumor resistance.


Current controversies in the endocrine therapy of advanced breast cancer.

The response of breast cancer to endocrine therapy depends on depriving the tumor cells of estrogen stimulation of cell division. This may be achieved by blocking estrogen synthesis in the body, using inhibitors of aromatase or by using drugs like tamoxifen, which interfere with the direct effects of estrogen on tumor cells. Current controversies relate to the mechanism for achieving more effective inhibition of estrogen synthesis and for the use of more specific antiestrogenic drugs.


Megestrol acetate in the treatment of metastatic carcinoma of the prostate.

Although current hormonal therapy of prostate cancer may not appear to have altered survival appreciably, there have been considerable changes that may significantly affect the future management of this disease. A number of new hormonal agents have been introduced that still require definition of their therapeutic efficacy. Megestrol acetate, a hormonal agent with multiple sites of action in androgen metabolism, has recently been investigated in the treatment of patients with metastatic and locally advanced disease, and in those patients whose disease progresses with other hormonal therapies. Megestrol acetate plus mini-dose diethylstilbestrol (DES) is associated with fewer side effects than standard-dose DES and has equivalent therapeutic efficacy in the treatment of patients with metastatic disease. In patients with locally advanced disease that may benefit from hormonal cytoreduction, megestrol acetate is effective and well tolerated. Megestrol acetate has a role in the palliation of patients with progressive disease despite initial hormonal therapy. Considerable controversy surrounds the therapy of carcinoma of the prostate; further studies are required to define optimal hormonal therapy.


Pathophysiology of cancer: hormonal and metabolic abnormalities.

Despite the development of advanced nutritional support technology, malnutrition remains a significant morbid and mortal complication of cancer. A number of metabolic abnormalities have been demonstrated in malnourished cancer patients, including increased protein breakdown, increased glucose production, increased lipolysis, hypogonadism in male patients, and insulin resistance. Previous studies conducted under metabolic ward conditions have demonstrated that metabolic abnormalities interfere with efforts at renutrition of patients with localized head and neck cancer. Efforts to correct these abnormalities by treatment with hydrazine sulfate, anabolic androgens, and insulinotropic drugs have been ineffective. An improved understanding of the pathophysiology of cancer malnutrition may lead to improved therapies of this vexing clinical problem.


Clinical aspects of nutrition in advanced cancer.

Nutritional assessments of our patients with disseminated malignancies have revealed that the incidences of reported anorexia, decreased food intake, and weight loss range between 49 and 64%. It is therefore essential that a planned approach to the nutritional needs of patients with advanced cancer be part of routine oncology care. Our first step is a clinical assessment of the patient's nutritional state and diet, and a determination of caloric and nutrient needs. The potential tools available to the oncologist in the management of the undernourished cancer patient are many and include dietary counseling, food supplements (which contain vitamins and other micronutrients), stimulation of appetite, enteral nutrition, total parenteral nutrition, or a combination of these. The dietitian can be an invaluable component of the cancer care team, both in the inpatient and outpatient settings. An understanding of the role of each intervention will enable the physician to use available resources rationally and efficiently.


Treatment of cancer weight loss in patients with advanced lung cancer.

Megestrol acetate, a semisynthetic gestagen, is effective not only for endocrine therapy of advanced breast carcinomas, but also in the treatment of cachectic patients with aggressive, hormone-independent tumors. Sixty-six patients with therapy-resistant, advanced bronchogenic carcinomas of different histologic types, who had lost more than 10% of their regular body weight within a prospective, randomized trial, were treated with megestrol acetate at a dose of either 160 or 480 mg/day for 3-4 months. The mean weight gain for all patients was 2.5 kg. Nearly all of them (80%) reported improved appetite and well-being. The mean weight gain in the group of patients receiving 480 mg/day was higher (3.0 kg) than in the group receiving 160 mg/day (2.0 kg).


Clinical management of anorexia and cachexia in patients with advanced cancer.

Cachexia occurs in the majority of cancer patients before death. It is the result of major metabolic changes produced by tumor-released substances as well as by cytokines and some endogenous peptides. The most significant clinical manifestation is profound anorexia. Aggressive parenteral nutrition has not been able to increase patient survival or produce any significant symptomatic improvement. Recent research, therefore, has focused on drugs that might result in symptomatic improvement, even if no significant nutritional changes are detected. Corticosteroids have been shown to increase appetite for a brief period of time, but they do not appear to improve caloric intake or nutritional status. In addition to appetite stimulation, corticosteroids also improve a number of other symptoms transiently. Progestational drugs have been found in a number of studies to increase appetite, caloric intake, and nutritional status. The most effective type and dose of progestational drugs have not been clearly established. Cyproheptadine, hydrazine sulfate, and cannabinoids have all been suggested to have beneficial effects on appetite; their effectiveness, however, needs to be confirmed in prospective, controlled trials. Some of these trials are currently under way. Current data suggest that megestrol acetate or other progestational agents could be useful--because of effects on not only appetite but also overall nutritional status--in patients who have profound anorexia as the main manifestation of cachexia, provided expected survival can be measured in weeks or months. In patients with shorter expected survival or those who have problems tolerating progestational drugs, a brief course of corticosteroids may provide short-term symptomatic effects. Future studies should focus on (1) improving understanding of both the pathophysiology of cancer cachexia and the interaction of some of the major syndromes of terminal cancer--e.g., pain, cachexia, and cognitive failure--and (2) characterizing the symptomatic effects of different drugs more completely.


Risks and benefits of various therapies for cancer anorexia.

A placebo-controlled, randomized trial assessed the activity, tolerance, and degree of weight gain and anorexia of two doses of megestrol acetate in patients with advanced cancer and cachexia. Patients received either low-dose (480 mg/day) or high-dose (960 mg/day) megestrol acetate or placebo for 8 weeks. As of March 1991, 91 patients had been randomized; 65 patients were evaluable. Median initial weight loss in all patients ranged from 13 to 16%. Further weight loss was seen in 13 of 17 patients in the placebo group, compared with 10 of 27 and 6 of 21 in the low- and high-dose megestrol acetate groups, respectively. Eight of 27 patients in the low-dose-group and 9 of 21 in the high-dose group gained weight, with a median of 3 and 4 kg, respectively. Beneficial effects of megestrol acetate were seen in 63 and 71% of the low- and high-dose groups, respectively, compared with 24% of the placebo group. Side effects of megestrol acetate were mild. No correlation between megestrol acetate dosage and weight gain was found, but there was a tendency for increased weight in more patients taking high-dose megestrol acetate.


Megestrol acetate for anorexia and cachexia.

Based on promising preliminary information obtained from uncontrolled pilot studies, several randomized, placebo-controlled, double-blind clinical trials have been launched to evaluate the effectiveness of megestrol acetate for the treatment of patients with anorexia and/or cachexia. The results of these studies have demonstrated that megestrol acetate can improve patients' appetites and promote nonfluid weight gain in some. They also suggest that megestrol acetate has antiemetic properties. Ongoing clinical trials are evaluating the dose-response relationship of megestrol acetate for these clinical problems and whether megestrol acetate will improve the survival of patients at risk for developing cancer anorexia/cachexia.


HIV-related cachexia: potential mechanisms and treatment.

Involuntary weight loss or wasting indicative of severe protein energy malnutrition is a frequent complication of acquired immune deficiency syndrome (AIDS). Malnutrition, with its associated adverse effects on immunocompetence, may contribute to the progression of AIDS itself. Since death from wasting is ultimately related to the magnitude of tissue depletion, restoration of body cell mass may enhance survival. The mechanism of weight loss in AIDS has not been clearly elucidated. The etiology is likely to be multifactorial, the result of interactions between decreased caloric intake, malabsorption, and alterations in energy expenditure secondary to hormonal and/or metabolic abnormalities. Although weight loss is occasionally reversible with treatment of underlying infections and/or easily identifiable and reversible causes, the majority of patients are not this fortunate. Enteral and parenteral nutrition, which are expensive, cumbersome, and potentially morbid, have been suggested by some as therapeutic options. Megestrol acetate, a synthetic, orally active progestational agent, has been reported to stimulate appetite and weight gain. Data regarding the use of megestrol acetate for the treatment of cachexia related to human immunodeficiency virus (HIV) infection demonstrate convincingly its effectiveness in treating many patients with HIV-related anorexia and cachexia.


High-dose megestrol acetate as third-line endocrine therapy for metastatic breast cancer.

High-dose megestrol acetate (800 mg/day) was administered to 34 patients as third-line endocrine therapy for metastatic breast cancer after progression on standard-dose megestrol acetate (160 mg/day). Among the 32 evaluable patients, no complete or partial response occurred. Ten patients remained stable and 22 progressed. No patients remained on study. Median time to progression was 2 months (range, 1-13 months). The use of high-dose megestrol acetate did not result in objective responses but may have been effective in delaying progression in one third of patients.


[The expression of the apolipoprotein A-1 gene in the early stages of human embryogenesis studied by hybridization in situ].

The apolipoprotein A-1 (apo A-1) gene expression at certain stages of human embryo development was studied using an in situ hybridization procedure. By the fifth week of development, the presence of apo A-1 mRNAs was detected in cells of different tissue types such as CNS rudiment, somite myotomes, ear vesicle, nasal placodes, lens, apical areas of the maxillary, mandibular and hyoid arcs, mesenchyme of limb buds, small intestine, mesonephros, genital ridge, and several types of blood cells. At later stages (weeks 8-9) the distribution of these mRNAs showed considerable changes. The high apo A-1 mRNA level was characteristic of liver cells, adrenals, kidney, neural tube and ganglia. These data suggest that changes in tissue-specific apo A-1 gene expression during human embryogenesis may be associated with the development of blood circulatory system and CNS.


[An immunohistochemical analysis of rat hippocampal antigens in early postnatal ontogeny by using monoclonal antibodies].

In order to study the molecular mechanisms of neurogenesis, monoclonal antibodies (MAbs) were produced against antigens of the developing rat hippocampus. MAb 3G7-F8 was used for immunohistochemical localization of the corresponding antigen of paraffin sections of the rat brain at days 0, 5, 14, and 21 of the postnatal development. In the hippocampus of newborn and 5-day-old rats, positive immunostaining was observed in the cytoplasm and proximal segments of processes of neurons located in granular, polymorph, and pyramidal layers, as well as in entorhinal cortex. In granule cell bodies and neurons of entorhinal cortex specific staining decreased by day 14 and disappeared by day 21 after birth, whereas neurons of pyramidal and polymorph layers remained immunopositive. Diffuse specific staining in the cerebellum was observed beginning from day 5 after birth in the Purkinje cell layer. On days 14-21 positive reaction was observed in Purkinje cell bodies and in the layer containing dendrites of Purkinje cells and parallel fibers. External and internal granular layers remained immunonegative. No specific staining was observed in other regions of the brain, as well as in the control slices. These data suggest that the antigen detected by the 3G7-F8 antibody is involved in the formation of the neuronal connections.


[Contractile proteins and nonerythroid spectrin in the oogenesis of the clawed toad].

Distribution of contractile proteins, actin and myosin, and spectrin was studied in oogenesis of X. laevis. These proteins are present already at the previtellogenic stages, where they are diffusely distributed. During vitellogenesis actin and myosin are distributed in the animal region in a fibril-like way, while in the vegetal one they are concentrated around the yolk platelets. In the mature oocyte, distribution of actin and myosin again becomes diffuse. Spectrin forms in the vitellogenic oocyte a network all over the cytoplasm, while in the full-grown oocyte it is localized mostly in the subcortex of the animal region and disappears during oocyte maturation. All these proteins are present in the nuclei of oocytes. Changes in distribution of actin, myosin and spectrin during oocyte maturation are discussed with reference to the cortical contractility, spatial distribution of yolk platelets and regional sensitivity to cytochalasin B.


[The nuclei of follicular cells do not control oocyte maturation in amphibians induced by gonadotropic hormones in vitro].

The inhibitory effect of actinomycin D (5 micrograms/ml) on maturation of the follicle-enclosed oocytes of Rana temporaria and Xenopus laevis induced in vitro by pituitary suspension and human chorionic gonadotropin, respectively, is not observed at low concentrations of the hormones. These data suggest that nuclei of the follicle cells are not involved in the control of the pituitary-dependent oocyte maturation in amphibians.


[The population kinetics of human male germ cells in the antenatal period of ontogeny].

Time course of development of male germ cells in man during prenatal development was studied quantitatively for the first time. For each period of the prenatal development the following characteristics were determined: relationship between the number of germ and somatic cells in spermatic cords, mitotic index for germ cells the intensity of their degeneration, average diameter of their nuclei, and the proportion of germ cells at the stage of preleptothene.


[Alcohol and the liver: ethanol metabolism and the pathomechanism of alcoholic liver damage].

Ethanol is oxidized in the liver by three different enzyme systems, namely by alcohol dehidrogenase (ADH), the microsomal ethanol oxidizing system and catalase. Alcohol also undergoes a first pass metabolism in the gastric mucosa due to alcohol dehydrogenase. This first pass metabolism of ethanol is decreased in the alcoholic, in the fasted state, in the elderly and during cimetidine therapy leading to elevated alcohol blood-concentrations. Ethanol toxicity is closely related to its metabolism in the liver. Ethanol oxidation by ADH generates reducing equivalents (NADH) and acetaldehyde (AA). The elevated NADH/NAD ratio results in alterations of the intermediary metabolism of lipids, carbohydrates, proteins, purines, hormones and porphyrins. Furthermore, NADH flavours free radical production. The ethanol-associated redox changes are pronounced in the perivenular zone, since this is the area of low oxygen tension and of high ADH activity. In addition to NADH, AA exerts striking toxic effects on the hepatocyte. AA binds to cellular proteins and membranes including the mitochondria, microtubules, glutathion and various enzymes. In addition, AA and lactate stimulate collagen production in fibroblasts. AA-adducts stimulate the production of antibodies against AA-epitopes and could thus aggravate the liver injury. Chronic ethanol consumption results also in the microsomal induction of a specific ethanol-inducible form of cytochrome P--450, the cytochrome P--450IIE1 with high affinity not only to ethanol but also to some drugs (acetaminophen), procarcinogens (nitrosamines) and industrial agents (carbon tetrachloride). The interaction between ethanol metabolism and the metabolism of these compounds including vitamin A may also contribute to hepatic toxicity, since the susceptibility of the alcoholic toward those compounds is enhanced.


[The dermatoglyphic analysis of polydactyly and the segregation of the morphogenetic fields of the fingers in man].

We studied dermatoglyphic imprints of patients with preaxial or postaxial polydactyly. Development of the additional finger is shown to be always accompanied by the corresponding finger triradius and basic line; postaxial polydactylies are characterized by the circular epidermal ridges around the rudiment of the sixth finger and the presence of the second additional triradius. On the basis of these data we suggest that major lines of the palm and finger triradii serve as landmarks denoting the borders of individual morphogenetic fields of fingers during their segregation from general morphogenetic field of the hand which is related to the polar system of coordinates.


[Significance of determination of squamous cell carcinoma antigen in the follow up of patients with cancer of the uterine cervix].

The serum concentrations of squamous cell carcinoma antigen (SCC) were determined in 59 patients with invasive carcinoma of the uterine cervix, in 21 patients with benign cervical diseases. Pretherapeutic SCC levels were elevated (> 2.5 ng/ml) in 63% (34/54) of the patients with squamous cervical carcinoma. 5 patients with adenomatosus cervical carcinoma had not elevated levels. In 21 patients with benign cervical diseases, 4.8% (1/21) of SCC values were above the normal range. There were no correlation of SCC levels and the degree of the histological differentiation or clinical stages. Serial SCC determinations revealed a correlation with the clinical course of disease in 72.2% (26/36) with squamous cervical carcinoma. Rising levels were always associated with progression. The SCC level increased before clinical signs of progression with a median lead time of 3 months. Patients with cervical adenocarcinoma for follow up CA 125 determination was recommended. The determination of SCC is a useful tool checking the patients, detecting early progression of squamous cervical tumor.


[Intracerebral transplants of embryonic nerve tissue result in the recovery of the ultrastructure of dystrophic neurons following hypoxia in adult rats].

Fragments of the intact rat embryonic brain cortex were transplanted into sensomotor cortex of adult Wistar rats subjected to hypoxia 26 days before surgery. Ultrastructure of the host brain was studied 4, 100, 130 days, 1.5, and 2 years after the operation using electron microscopy. The observed regeneration of ultrastructure of the host's neurons appears to be due to either recovery or de novo formation of the organelles.


[False positive results of exercise tests with thallium myocardial scintigraphy].

The authors have examined supposed causes of positive Th-201 stress scintigraphy. Patients were divided into two groups according to the presence of exercise-induced transitory hypoperfusion or in absence of it. In both groups negative coronarograms were verified. Results of two groups were compared according to parameters of the left ventricle function, pulmonary pressure, to the presence of disorder of system stimulus-conduction (LBBB, RBBB), mitral prolapsus, foramen ovale apertum, anomalous coronary anatomy, level of load during exercise, body weight, heart volume-index. Significant difference was found in disorder of left ventricle motion, ejection fraction and in comprehensive value of left ventricle function (F1).


[The three-dimensional structure of the tunica intima and tunica media of the human fetal aorta studied by a new method].

A new method is developed for revealing the latent surfaces in the structure of organs by scanning electronic microscopy. The method is based on the treatment of specimens with potassium ethoxide until cells start to appear in the dissociating solution. Using this method, thoracic aorta of nine human fetuses at the stage of 20-28 weeks was studied. Subendothelial intima and media of human fetal aorta contain smooth muscle cells differing by their arrangement, shape and surface microrelief. The intima cells are arranged in a mosaic pattern formed of single cells or cell clusters. By means of cell processes they are connected with each other, as well as with endothelial and smooth muscle cells of the media. Smooth muscle cells in the inner part of the media also have processes and form an open network. Part of the cells penetrate the intima through pores of the inner elastic membrane. In the deeper layers of the media, laterally adjoining spindle-shaped smooth muscle cells are found. It is suggested that the observed cell polymorphism is due mostly to penetration of the media smooth muscle cells into subendothelium and modification of their shape under the effect of the microenvironment.


[Incidence of intrauterine growth retardation and its significance in perinatology in Hungary].

The authors have examined the incidence of intrauterine growth retardation at a country level for the first time both in local and international aspect. Using the nomenclature of NDN-system they have found the frequency of WL-(proportional) retardation 7.95% and that of N-(disproportional) retardation 5.99%. They show incidence of retardation from county to county, as well. Examining the correlation between retardation and perinatal mortality they've stated that while the frequency of still-birth among the average developed and proportionally nutrified fetus is 0.34% and the frequency of perinatal mortality among them is 1.23%, that of the N-retardated newborns is 0.88% and 2.08%, respectively, and that of the WL-retarded is higher than the latter: 2.03% and 3.90% respectively. Regarding the incidence and mortality ratio among the two types of retardation the difference is significant.


[Pancreatic cystic tumor misdiagnosed as pseudocyst: lessons from an intraoperative error].

A 62-year old woman presented a fistula due to a previously made cystojejunostomy performed because of a small pancreatic lesion considered as a pseudocyst. This intervention was performed in another institute. In the course of the exploration the authors resected the whole mass with the Roux-en-Y loop, and with the spleen. The histological examination proved a cystadenocarcinoma of the pancreas. There are discussed the problems of the intraoperative diagnosis of pancreatic lesions, and the possibilities of the reduction of mistakes. The authors emphasize the experience of the surgeon--which is the most important factor of the cure--and the importance of the fine needle biopsy.


Proviral insertions near cyclin D1 in mouse lymphomas: a parallel for BCL1 translocations in human B-cell neoplasms.

By isolating genomic DNA clones that encompass the mouse Cyl-1 (cyclin D1) locus, we have identified a putative CpG island close to the 5' end of the gene. Pulsed-field gel electrophoresis with probes derived from either the 5' or 3' side of the CpG island established physical linkage to two independent markers on mouse chromosome 7, in a region that is syntenic with human chromosome 11q13. On the 3' side, Cyl-1 is approximately 75 kb from Hst-1 and Int-2, although there is an additional CpG island in the intervening DNA, while on the 5' side, Cyl-1 is less than 300 kb from Fis-1, an integration site for Friend murine leukaemia virus. As there is no intervening CpG island, proviral insertions at Fis-1 could influence the expression of Cyl-1 and we describe two virally induced tumours in which this appears to be the case. The data suggest that proviral insertions near Cyl-1 in mouse lymphomas are functionally equivalent to the BCL1 translocations that activate cyclin D1 expression in human B-cell malignancies.


T-cell acute lymphoblastic lymphoma induced in transgenic mice by the RBTN1 and RBTN2 LIM-domain genes.

Two members of the RBTN gene family, RBTN1/Ttg-1 and RBTN2/Ttg-2, were found by their association with T-cell tumour-specific chromosomal translocations and are thought to be involved in the aetiology of such T-cell tumours. Here a transgenic mouse model is described in which T-cell tumours are induced by the presence of RBTN1 and RBTN2 transgenes that direct expression in thymus-derived cells. The latency period for lymphoid tumour appearance is variable, and tumours occur in a small proportion of transgenic animals that develop T-cell acute lymphoblastic malignancies. No significant increase in the rate of tumour development was observed in RBTN1 transgenic mice infected with Moloney murine leukaemia virus, nor did tumours arise in mice bearing a construct in which RBTN1 was expressed from the insulin transcriptional promoter. These data, which provide formal proof of the oncogenic activity of these genes, suggest that aberrant expression of transcription factor genes, such as RBTN1 and RBTN2, functions in tumour aetiology by disturbing some aspect of T-cell differentiation.


Brain tumours and lymphomas in transgenic mice that carry HTLV-I LTR/c-myc and Ig/tax genes.

The human T-cell leukemia virus type 1 (HTLV-I) is associated with adult CD4+ T-cell leukemia (ATL) and tropical spastic paraparesis (TSP). In as much as only a small percentage of individuals infected with HTLV-I develop either disease, we set out to model a genetic partner for this virus in an effort to understand and possibly reproduce its pathophysiology. To this end we have developed a binary set of transgenic mice, one bearing the relatively inactive HTLV-I long terminal repeat (LTR) positioned to drive the c-myc oncogene and another bearing a fusion transgene consisting of the immunoglobulin promoter/enhancer driving the gene for the HTLV-I transcription activator, tax. Alone, the tax construct, though expressed in the thymus, spleen, lung and brain, has no deleterious effect. Alone, the HTLV-I LTR/c-myc construct is expressed at very low levels in lymphoid cells and occasionally induces lymphomas in older animals. When these two transgenic lines are mated, bigenic offspring harboring both transgenes exhibit dramatic tumor formation. As in the human, these animals develop CD4+ T-cell lymphomas, but they also develop central nervous system tumors by 25-90 days of age. The syndrome, which is 100% penetrant and lethal, provides an animal model for adult T-cell lymphoma and a source of cultured cells of neurogenic origin.


A novel NF-kappa B element within exon 1 of the murine c-myc gene.

We have mapped a site within exon 1 of the murine c-myc gene that forms a variety of complexes with nuclear proteins derived from the murine WEHI 231 B-lymphoma cell line in exponential growth that are altered following treatment with phorbol ester, when transcription of this gene is reduced [Levine, R.A., McCormack, J.E., Buckler, A.J. & Sonenshein, G.E. (1986). Mol. Cell Biol., 6, 4112-4116]. This site, located at +440 to +459 bp relative to the P1 promoter, contains an NK-kappa B-like binding element. The sequence of this element, AGGGAATTTTT, is unusual in that the stretch of pyrimidines is entirely T residues. Binding of NF-kappa B protein was demonstrated by oligonucleotide competition, induction of binding upon 70Z/3 pre-B- to B-cell differentiation, response to GTP in the binding reaction, reduction of binding upon addition of I kappa B protein and uv cross-linking analysis. Functional activity of this internal regulatory element (IRE) was demonstrated in transfection assays using chloramphenicol acetyl transferase (CAT) reporter constructs containing multimerized copies of the IRE driving a heterologous promoter. Mutation of the IRE within the context of the c-myc promoter prevented NF-kappa B-mediated induction of transcription of this oncogene. Thus additional NF-kappa B elements may be defined by this new sequence.


c-myc and p53 gene expression in the differentiation of temperature-sensitive mutants of teratocarcinoma F9 cells.

We have previously reported the isolation of several temperature-sensitive (ts) mutants of F9 cells. Further investigations showed that some mutants were induced to differentiate at non-permissive temperature of cell growth, accompanied by changes in the expression of various genes, whereas others were not. During the differentiation induced by shifting up to the non-permissive temperature, a rapid and transient decrease in both c-myc and p53 mRNA levels and rapid induction of c-jun mRNA were observed. These changes were specific in differentiation-inducible mutants and were not observed in a non-inducible mutant. In both types of mutants, the level of c-myc mRNA decreased in association with growth retardation at the non-permissive temperature. The p53 mRNA, however, showed specific increase in the differentiation-inducible ts mutants. These observations suggest distinct roles for p53 and c-myc from proliferation to differentiation in teratocarcinoma stem cells.


Molecular characterization and structural organization of D-elg, an ets proto-oncogene-related gene of Drosophila.

We have continued the molecular analysis of D-elg, a member of the Drosophila ets gene family. Based on the characterization of cDNA and genomic sequences, the D-elg gene contains five exons and four introns and produces a mRNA with an open reading frame of 464 amino acids. Consistent with this analysis, in vitro translation of a near full-length D-elg cRNA yields a protein with a molecular weight of approximately 56 kDa. D-elg shows significant homology to other ets proteins in the amino-terminal A domain and strong homology in the carboxy-terminal ETS domain. The D-elg protein is most similar to the alpha-subunit of the mouse GA-binding protein.


Characterization of activated and normal mouse Mos gene in murine 3T3 cells.

We have characterized the mouse Mos proto-oncogene product, pp39Mos, in murine fibroblasts. When expressed in NIH3T3 cells under the influence of the long terminal repeat regulatory element from Moloney murine sarcoma virus [NIH(pTS-1) cells], the Mos protein was present in low levels and had a half-life of about 30 min. In extracts from NIH(pTS-1) cells, we detected additional forms of Mos protein that apparently arose from internal initiation codons (p24Mos and p29Mos) or from upstream non-AUG initiation codons (p42Mos and p44Mos). The Mos protein was found to exist in these cells as a phosphoprotein, pp39Mos, and, when immunoprecipitated with an antiserum specific for the Mos N-terminus [anti-Mos(6-24)], had autophosphorylating kinase activity. We found that anti-Mos(6-24) also detected non-Mos protein kinase activity and non-Mos phosphoproteins in addition to p39Mos. We present evidence, on both the RNA and protein levels, that non-transformed mouse 3T3 cells do not express endogenous Mos.


Increased expression of the negative growth factor, galactoside-binding protein, gene in transformed thyroid cells and in human thyroid carcinomas.

Murine beta-galactoside-binding protein has been shown to be a cell growth regulatory molecule and a cytostatic factor. We analysed the beta-galactoside-binding protein gene expression in a thyroid cell system including two normal cell lines (FRTL-5 and PC Cl 3) and the same cells transfected by several oncogenes that induce different degrees of malignancy and differentiation. We show that beta-galactoside-binding protein mRNA levels correlate with the expression of the malignant phenotype. Run-on experiments suggest that a transcriptional effect accounts at least in part for such a difference. We also show that the beta-galactoside-binding protein gene expression is increased in most human papillary thyroid carcinomas compared with normal thyroid.


DNA amplification at 11q13.5-q14 in human breast cancer.

Band q13 of chromosome 11 is frequently amplified in human breast cancers, but the gene(s) responsible for the emergence of this amplicon remain(s) elusive as yet. As a tribute to the complexity of the amplification events involving 11q13 sequences in human breast cancer, we have now studied a more telomeric region at 11q13.5-q14 defined by a new transcription unit, D11S833E. We have observed that amplicons present in cell lines and primary tumors amplified for both BCL1 and D11S833E could be interrupted between these two loci. Such discontinuities were demonstrated by using a probe for the KRN1 gene, which we have localized between the BCL1/FGF4 region and D11S833E. In fact, KRN1 was not present in 4 out of 10 amplicons bearing both BCL1 and D11S833E. Furthermore, we have observed tumors in which D11S833E could be amplified in the absence of amplification of other known markers of 11q13. Therefore, D11S833E defines a new and independent amplification unit in this region.


Rearrangement of the human tre oncogene by homologous recombination between Alu repeats of nucleotide sequences from two different chromosomes.

The rearranged region of the tre oncogene originating from chromosomes 5q23q31 and 18q12 was cloned from tumor genomic DNA, sequenced and aligned with wild-type sequences cloned from a normal human genomic library. In the breakpoint region each wild-type sequence contained two Alu repeats. The recombination occurred between the 3'-most Alu from chromosome 5 and the 5'-most Alu from chromosome 18 and, consequently, resulted in a hybrid Alu flanked with one Alu on either side. The recombinant joint was located to a 20-bp homology region in left arms of the Alu repeats involved in recombination. The same homology region was identified in the hybrid Alu of the rearranged tre. At its 5' extremity the homology region overlaps the B box of Alu-borne RNA polymerase III promoter. The 100% identity score in the region of homology suggests that the recombination process was conservative and not error prone.


Inactivation of the murine N-ras gene by gene targeting.

The mammalian ras genes have been implicated in the genesis of a wide variety of tumors. Although it is likely that they play an essential role in signal transduction, their specific function is still unknown. To initiate a genetic analysis of the ras genes in mammals we inactivated the N-ras gene in murine embryonic stem cells by gene targeting. The frequency of integration at the N-ras locus of our targeting vector being low (of the order of 1/5000 transfectants), we used a positive/negative selection followed by an analysis of individual colonies in order to minimize the in vitro manipulation of the embryonic stem cells. Using this approach, we isolated two clones of ES cells with one inactivated N-ras allele. These cells have no distinctive phenotype either in vitro or in vivo in chimeric mice.


Alternative splicing in the human lck gene leads to the deletion of exon 1' and results in a new type II lck transcript.

In this study we describe a new lck mRNA transcribed from the type II promoter, resulting from alternative splicing, in the deletion of exon 1' which comprises the AUG initiation codon. In this type IIB lck transcript, as a consequence of exon 1' deletion, a new AUG codon in exon 1, located 35 nt upstream from the regular initiation codon in exon 1', and normally out of frame, is now in frame with exon 2 and the following lck coding sequence. In this type IIB lck transcript, 10 residues encoded by exon 1 from the new AUG codon replace the first 35 residues encoded by exon 1'. Strikingly, in this new N-terminal amino acid sequence, a glycine residue, needed for myristylation and anchorage in the plasma membrane, is present at position 2. This alternative splicing has been observed in T-cell lines, normal mature T cells and in peripheral blood lymphocytes from different leukemic patients. Different ratios of regular type IIA lck mRNA (with exon 1') to spliced type IIB lck mRNA (without exon 1') have been observed depending on the patient.


p53 and RAS gene mutations in multiple myeloma.

We analysed genomic DNA from 30 patients with multiple myeloma (MM), searching for alterations in the p53 and RAS genes by a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. Mutations in the p53 gene were observed in 20% (6 out of 30) of the patients, and were located in conserved sequence blocks within exons 5 and 7. These were single-nucleotide substitutions and consisted predominantly (4/6) of G:C to A:T transitions. Of the six patients with a mutated p53 gene, four were in the terminal phase of the disease. RAS gene mutations were found more frequently since they occurred in 47% (14 out of 30) of the patients. Mutations consisted of single-nucleotide substitutions, located in codons 12, 13 and 61 of either K- or N-RAS, to the exclusion of H-RAS. Moreover, one patient bore two simultaneous mutations, affecting simultaneously the K- and the N-RAS genes. RAS gene mutations were more frequently observed in patients with fulminating disease (10/15, 67%) than in patients with less aggressive forms of the disease (4/15, 26%). We also analysed genomic DNAs from 10 human myeloma cell lines, of which two bore mutations affecting codon 12 of the K-RAS gene, and one codon 12 of the N-RAS gene. The first two cell lines were obtained from freshly explanted tumor cells in which we observed identical mutations. Results presented here show that activating mutations in the RAS genes are, in MM, more frequent than those affecting the p53 gene and suggest that both events are related to terminal phases of the disease.


The amino acid sequence of a mammalian MAP kinase kinase.

The amino acid sequence of the dual specificity mitogen-activated protein kinase kinase (MAPKK) has been determined by cDNA cloning and amino acid sequencing. MAPKK (393 residues, Mr 43,330) is a new member of the protein kinase subclass that comprises byr1 and STE7 that are involved in pheromone dependent signal transduction in yeast, wis1 a mitotic regulator in S. pombe and PBS2, which confers antibiotic resistance in S. cerevisiae.


The anterior cruciate ligament-deficient knee: a diagnostic and therapeutic algorithm.

The anterior cruciate ligament (ACL) of the knee is a common site of sports-related injury. The natural history of chronic instability, reinjury, and further intra-articular damage is well documented. Appropriate management of ACL insufficiency must be based upon a knowledge of the available diagnostic and therapeutic options, their indications, and their effectiveness. At the same time, the treatment plan must be tailored to the demands and expectations of the individual patient.


Meniscal tears: diagnosis, evaluation, and treatment.

Arthroscopic surgery for treatment of pathologic meniscal lesions has become the most commonly performed orthopaedic procedure in the United States. This article reviews the diagnosis, evaluation, and subsequent treatment of meniscal tears in the knee.


Myositis ossificans traumatica.

Myositis ossificans is a benign condition of heterotopic bone formation. It is a generalized term for four separate clinical entities. Although not a rare disease process, diagnosis often remains difficult. This article reviews the various forms of myositis ossificans, as well as the pathology, diagnostic tests, and treatment options.


New outpatient treatment of recurrent patellar dislocations.

From December 1987 through February 1991, outpatient arthroscopic procedures were done on patients experiencing recurrent dislocations of the patella. A modified Emslie-Trillat procedure was used. Twenty-four patients (27 knees) were available for follow-up. Using the criteria of Cox, excellent results were found in 78% (21 knees), whereas 18% (5 knees) were rated as good. Only 4% (1 knee) was considered fair, and none were rated poor. Most patients (96%) showed improvement. No patient suffered a postoperative dislocation. Based upon the findings of this study, it is concluded that this procedure is an effective method of treatment for patients having recurrent dislocations of the patella.


Difficult reductions in traumatic patellar dislocation.

A traumatic lateral dislocation with internal rotation of the patella can be unreducible using conventional maneuvers. These unusual dislocations may involve locking of the patella on the lateral femoral condyle. In these cases, reduction can be achieved by applying a downward force to the lateral aspect of the patella, which reduces the rotational deformity and unlocks the medial patellar facet.


Brachial plexus injury with erect dislocation of the shoulder.

Luxatio erecta, or inferior dislocation of the glenohumeral joint, is an extremely uncommon variety of shoulder dislocation. Several types of neurovascular injuries may be associated with luxatio erecta. Concomitant fracture of the coracoid, clavicle, acromion, greater tuberosity, and humeral head may also be noted. A case of luxatio erecta associated with a fracture of the greater tuberosity and transient mixed brachial plexus injuries is presented.


A unicompartmental knee replacement is not "half a total knee": five major differences.

Unicompartmental knee replacement surgery has known uneven success. It fell out of favor in the late 1970s as it was gradually replaced by total knee arthroplasty. The failures of unicompartmental replacements were due partly to the implants themselves but largely to incomplete knowledge of the principles and techniques associated with the unicompartmental knee replacement procedure. As newer implants become available, it is easy to focus on the technology at the expense of basic principles. The authors elaborate on five key principles that differentiate unicompartmental knee replacement from total knee arthroplasty.


A 22-year-old man with a swollen left hand after a motor vehicle accident.

The following case is presented to illustrate roentgenographic and clinical findings of a condition of interest to the orthopaedic surgeon and emergency room physician. The initial history, physical examination, and pertinent roentgenographic findings are found on this page. The final clinical diagnosis is presented on the next page.


Cytotoxic activity of 13-hydroxyoctadecadienoic acid against Toxoplasma gondii.

Recent data indicate that platelets may play an important role in the host defence against Toxoplasma gondii infections. T. gondii-stimulated human platelets release thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE) from arachidonic acid and 13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid (Yong et al. 1991; Henderson et al. 1992). We have previously demonstrated that the eicosanoid TXA2 has potent cytotoxic activity against T. gondii trophozoites (Yong et al. 1991). In this study, we examined whether 12-HETE, 13-HODE, and linoleic acid also have toxoplasmacidal activity. 13-HODE at concentrations > or = 10(-8) M rapidly induced cytotoxic changes in T. gondii. Ultrastructural changes induced by 13-HODE in T. gondii included an initial leakage of cytoplasmic contents into a space between the inner and outer parasite bilayer membrane units which was followed by intracellular vacuolation and loss of cytoplasmic contents. In contrast, linoleic acid and 12-HETE lacked toxoplasmacidal activity at 10(-10)-10(-6) M concentrations. These data indicate that 13-HODE, a product of linoleic acid metabolism, has potent cytotoxic activity against T. gondii; this toxoplasmacidal activity may be important in the inflammatory response to this pathogen.


Plasmodium berghei and Plasmodium chabaudi chabaudi: development of simple in vitro erythrocyte invasion assays.

Erythrocyte invasion assays are described for two species of rodent malaria, namely Plasmodium berghei and P.c. chabaudi. These invasion assays are simple, are carried out using a candle jar and allow a number of assays to be performed simultaneously. Our results demonstrate that both rodent malaria species show an in vitro preference for reticulocytes although the preference of P. c. chabaudi for these cells is not as marked as that of P. berghei. The details of our invasion assays and our results obtained are discussed.


Assessment of parasite population dynamics in mixed infections of rodent plasmodia.

Cloned lines of the four rodent Plasmodium species can be differentiated by the RFLP pattern generated following Southern blotting and probing with PCsv4.1, a probe derived from a P. chabaudi chabaudi genomic library. Groups of CBA/Ca mice were inoculated simultaneously with cloned lines from two parasite species or strains. Six mixed species and three mixed strain infections using rodent malaria lines were initiated. The composition of the parasite population in each group was determined qualitatively and semi-quantitatively by analysis of the DNA purified from daily blood samples, thereby providing a dynamic representation of each mixed infection. Effects on the course of parasitaemias are presented and discussed.


Selection of specific genotypes of Giardia intestinalis by growth in vitro and in vivo.

This study examined whether allelic changes observed when clinical isolates of Giardia intestinalis made in suckling mice were adapted to in vitro growth occurred as a result of gene switching (alternate isoenzymes) or through selection of organisms with different genotypes from mixed infections. Samples were compared electrophoretically at 20 enzyme loci. Marked allelic differences were detected between the uncloned clinical isolates grown in mice and the axenic cultures established from them. Furthermore, the allelic profiles of the uncloned isolates changed during the course of in vivo or in vitro growth. In contrast, all clones produced from each isolate retained identical allelic profiles, regardless of whether they were grown in vivo or in vitro. These findings argue against gene switching as an explanation for the observed allozyme changes and support preferential selection of organisms with specific genotypes by growth conditions. The data indicate the presence of at least 2 and possibly up to 4 distinct genotypes within each clinical isolate. The genetic differences detected between clinical isolates in suckling mice were of similar magnitude to those that separate different axenic isolates of G. intestinalis into cryptic species. Conversely, the genetic differences between the isolates were limited when sampled after establishment in vitro. These findings have significant implications for research on Giardia and other medically important parasites and raise the possibility that culture may exert a similar selective bias on the genotypes isolated from infections with other parasitic protozoa.


Proteinases of Trichomonas vaginalis: antibody response in patients with urogenital trichomoniasis.

Immunoprecipitation combined with electrophoresis in gelatin-polyacrylamide gels was successfully used for detection of antibodies against numerous proteinases of Trichomonas vaginalis in infected patients. The method proved to be highly specific as anti-proteinase antibodies were absent in women with negative cultivation of T. vaginalis and no history of trichomoniasis. Sera of 71% and vaginal washes of 86% patients with trichomoniasis were positive for these antibodies. In vaginal washes, but not in sera, antibodies were partly complexed with proteinases, possibly of trichomonad origin. It was also shown that serum antibodies as well as local anti-proteinase antibodies persisted for weeks after patients had been cured.


Impairment of growth of Leishmania donovani by Trypanosoma brucei during co-culture.

Cells of Leishmania donovani in co-culture with Trypanosoma brucei, were severely affected in their growth, resulting in swelling and subsequent lysis. Similar effects were also observed when Crithidia luciliae or Phytomonas sp. were co-cultured with T. brucei. Direct contact between the cells under investigation and T. brucei was necessary because T. brucei did not hamper the growth of the other trypanosomatids, when separated by a filter with 0.2 microns pore size. Examination of this phenomenon at the ultrastructural level, in a co-culture of L. donovani and T. brucei, suggests that the plasma membrane permeability is increased in the former, as a result of a close cellular contact between the two cell types.


Trichobilharzia ocellata infections in its snail host Lymnaea stagnalis: an in vitro study showing direct and indirect effects on the snail internal defence system, via the host central nervous system.

In this in vitro study we investigated whether previously described in vivo plasma-associated effects, that occurred in the period shortly after penetration of Trichobilharzia ocellata into the snail host Lymnaea stagnalis (1.5-72 h post-exposure; p.e.) were direct and/or indirect effects of parasite-derived factor(s). It was investigated whether the effect is mediated by the central nervous system (CNS) of the host. Phagocytic activity of the haemocytes was taken as a parameter for the activity of internal defence of the host. A number of preliminary experiments were performed. When the supernatant of in vitro cultured parasites (33 h; corresponding with their developmental stage in vivo when plasma-associated activation was found) was applied directly to monolayers of haemocytes, it appeared to enhance their phagocytic activity. No direct effect, however, was found with a supernatant of parasites cultured for a longer period of time (72 h; when, in vivo, a plasma-associated suppression was found). In this case, indirect suppression was detected: the parasites appeared to have released a factor that induced the CNS of the host to release material suppressing the activity of the internal defence system of the host. To date the nature of this factor is unknown.


Analysis of the complete sequence of a muscle calcium-binding protein of Schistosoma mansoni.

The complete sequence of the cDNA encoding a 20 kDa calcium-binding protein of Schistosoma mansoni (Sm20) has been determined. The predicted amino acid sequence contains 4 EF hand domains but examination of the predicted secondary structure of Sm20, together with the specific residues in each calcium-binding domain, suggests that only 1 EF hand (domain IV) is functional. Sm20 is most homologous to calmodulin, troponin C and the regulatory light-chain of myosin, particularly those of invertebrates. However, troponin C and the regulatory light-chain of myosin can be distinguished from Sm20 by size and by their differential levels of expression during the life-cycle. Sm20 also appears to be distinct from calmodulin but may be functionally equivalent to the soluble sarcoplasmic calcium-binding proteins of molluscs and crustacea which may act as a reservoir for calcium in muscle. Sm20 is encoded by a small multi-gene family whose members are clustered within a 15 kb region of the genome. A 20 kDa antigen, cross-reactive with Sm20, is expressed in Schistosoma haematobium, Fasciola hepatica and Paragonomus mexicanus.


Theileria annulata-infected cells produce abundant proteases whose activity is reduced by long-term cell culture.

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS-PAGE. The infected cells produced a number of extra activities at pH 8.0 in the presence of Ca2+. Calcium was found to enhance the activities but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.


Antibodies against somatic antigens and excreted/secreted products of Brugia pahangi in rats with patent and non-patent infections.

The humoral responses of Sprague-Dawley rats infected with Brugia pahangi were examined for up to 6 months after infection by ELISA, immunoblotting, and IFAT. In 2 experiments, 50% and 62.5% of rats developed patent, microfilaraemic infections. Mean adult worm burdens at autopsy were approximately 2% of the inoculum, and only patent rats yielded living adult worms. IgG antibody levels against crude somatic extracts (CSE) of all parasite stages and against adult excreted/secreted (ES) products were significantly higher in patent than non-patent rats. Both patent and non-patent rats produced anti-microfilarial surface antibody, as revealed by immunofluorescence. Immunostaining of Western blots by early infection sera showed no consistent difference in recognition of infective larval (L3) antigenic components by IgG or IgM antibody between eventually-patent and eventually-non-patent rats. By 26 weeks, however, patent rats recognized more components. The data suggest that antibodies against L3, adult, and microfilarial somatic antigens, ES antigens and microfilarial surface antigens do not correlate with the subsequent development of microfilaraemia in any individual rat.


Host strain, H-2 genotype and immunocompetence do not affect the survival or development of Onchocerca lienalis infective larvae implanted within micropore chambers into mice or rats.

The survival, growth and development of Onchocerca lienalis 3rd-stage (L3) larvae implanted into mice within micropore chambers has been studied with a view to developing a vaccination model for studies of protective immunity in onchocerciasis. The influence of host genetics on worm recoveries and development (growth and moulting rate) was analysed in a panel of inbred mice (CBA, BALB/c, DBA/2, SJL, 129J, C57BL/10 (B10), C3H/He and NIH), together with mice of BALB and B10 backgrounds with different major histocompatibility complex (H-2) genes (BALB/c, BALB.K, BALB.B and B10, B10.D2/n, B10.BR, B10.S). Parasite recoveries and development were similar in all mouse genotypes tested. They were unaffected by procedures designed to block or modulate phagocytic cell function with carbon or carrageenan, or to suppress inflammation by treatment with hydrocortisone acetate. A comparison of chambers sealed with membranes designed to admit (5.0 microns pore size) or exclude (0.2 microns pore size) host cells demonstrated no effect on the percentage recovery of living larvae, although dead larvae were more frequently retrieved when cells were excluded. Recoveries and rates of development of larvae implanted into immunodeficient scid mice and athymic Hooded rats were similar to those recorded in immunocompetent controls. We conclude that host genetic factors and immunocompetence are not significant determinants of survival, growth or development of O. lienalis larvae implanted within micropore chambers into naive mice. Despite its limitations, the use of this system merits further investigation as an approach to the study of protective immunity against developing larvae in onchocerciasis.


The effects of tumour necrosis factor on host-parasite relations in murine Mesocestoides corti (Cestoda) infection.

The regulatory role of tumour necrosis factor (TNF) was investigated in murine infection with tetrathyridia of Mesocestoides corti. Recombinant TNF alpha reduced macrophage larvicidal activity in vitro. M. corti primed mice for TNF release in response to bacterial lipopolysaccharide (LPS) in vivo. TNF activity was amplified 100-fold at 14 days post-infection (p.i.), with a further rise at day 28 p.i. Maximal inflammatory reaction was observed histologically in the liver at the height of TNF activity. Hepatic necrosis was located within inflammatory foci, but not within the vicinity of the parasite itself, suggesting that TNF may contribute to the pathogenesis of infection. Peritoneal cells from infected mice, when stimulated with tetrathyridia in vitro, showed a 4-fold increase in TNF alpha activity at day 14 p.i. However, when peritoneal cells were stimulated with LPS in vitro, a marked increase in TNF alpha secretion was observed at 2 months post-infection followed by a slow decline. It is suggested that impaired macrophage effector function, previously attributed to endogenous endotoxin, which gains access to peritoneal macrophages through an inability of the liver to detoxify endotoxin, may be mediated through TNF alpha.


Epidemiology of nematode infections of Soay sheep (Ovis aries L.) on St Kilda.

The epidemiology of nematode infections of Soay sheep on the island of St Kilda over a period of 2 years (August 1988-August 1990) spanning a host population crash is described. Infective larvae (L3) levels on pasture were high (2422 +/- 365 L3/kg D.M. grass in midsummer 1988) when host population density was high, decreasing after the sheep population declined by 70% in early 1989 (601 +/- 14 L3/kg D.M. in midsummer 1989). The availability of infective larvae to sheep increased during the winter of 1988-1989, probably as a result of concentration of existing larvae on grass as vegetation was destroyed by bad weather and overgrazing. Increased availability of pre-parasitic stages was accompanied by a marked increased in faecal egg counts from sheep of all ages and both sexes. Prevalence and intensity of infection (faecal egg counts) were higher in males than females throughout the 2-year study (chi 2 = 208.3, P < 0.005 and F1, 2000 = 304, P < 0.001 respectively), except during the lambing periods, and decreased with age in both sexes. Changes in prevalence and intensity of strongyle infections were associated with changes in host population density. Prevalence and intensity of Dictyocaulus filaria larvae in faeces increased during the host population crash. Infection intensity decreased with age (F1, 203 = 44.02, P < 0.001) and was higher in males than females (F1, 203 = 13.45, P < 0.001).


The role of nematode parasites in Soay sheep (Ovis aries L.) mortality during a population crash.

In early 1989, two-thirds of the Soay sheep population on St Kilda died over 12 weeks. Post-mortem examinations revealed emaciated carcasses and considerable nematode burdens, with protein-energy malnutrition as the probable cause of death. Haematological and blood biochemical changes in the sheep, as well as fecundity of gastrointestinal nematodes, suggested the hosts were immunosuppressed. In parallel, laboratory experiments in which Soay sheep on a high plane of nutrition were artificially infected with Ostertagia circumcincta, showed no clinical signs or mortality when supporting worm burdens similar to those recorded in dead sheep on St Kilda. Anthelmintic treatment of a group of animals increased daily survival rates in ewes and male lambs, although treated animals became re-infected as the 'crash' progressed. It is suggested that parasites contribute to mortality in malnourished hosts, exacerbating the effects of food shortage.


Neuropeptide F (Moniezia expansa): localization and characterization using specific antisera.

Immunocytochemical techniques used in conjunction with confocal scanning laser microscopy (CSLM) and electron microscopy have been used to demonstrate, for the first time, the distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF) in the cestode, Moniezia expansa. Antisera were raised to intact NPF(1-39) and to the C-terminal decapeptide of NPF(30-39). These antisera were characterized and validated for use in both immunocytochemistry and radioimmunoassay (RIA). NPF immunoreactivity (IR) was detected using both antisera throughout all of the major components of the central and peripheral nervous systems of the worm. The pattern of NPF-IR was found to mirror the IR obtained using a C-terminally directed pancreatic polypeptide (PP) antiserum and FMRFamide antisera; blocking studies using these antisera revealed that FMRFamide and PP antisera cross-react with NPF(M. expansa). RIA of acid-alcohol extracts of the worm measured 114 ng/g using the C-terminal NPF antiserum and 56 ng/g using the whole-molecule-directed antiserum. While the C-terminally-directed NPF antiserum cross-reacts with NPF-related peptides from other invertebrates, the whole-molecule-directed NPF antiserum is specific for NPF(M. expansa). The C-terminal NPF antiserum has potential for use in the identification and purification of NPF analogues from other platyhelminth parasites.


Population-based study of measles and measles immunization in human immunodeficiency virus-infected children.

This study reports the course of measles and results of measles immunization in a cohort of human immunodeficiency virus-infected children. Six cases of measles were identified. All had typical clinical manifestations, 5 of 6 developed pneumonia and 3 of 6 died. A measles intervention program consisting of serologic screening and active immunization (measles-mumps-rubella (MMR)) was instituted in 1990. Among 127 children with data available for analysis (mean age, 6.7 years), only 35% had documentation of prior immunization with MMR. Among 80 children who had preimmunization measles serology reported, 56% were measles antibody-negative and 40% were antibody-positive; following intervention 36% remained measles antibody-negative. Six children lost measles antibody over time. MMR nonresponders had lower CD4 lymphocyte counts (303 +/- 394) compared with responders (865 +/- 677; P = 0.0058). Measles is a potentially fatal illness in human immunodeficiency virus-infected children. Prevention strategies are limited by low rates of age-appropriate MMR immunization, poor antibody responses to MMR in older human immunodeficiency virus-infected children and seroreversion.


Infectious risks of Broviac catheters in children with neoplastic diseases: a matched pairs analysis.

In the present study the complication rate of Broviac catheters in the therapy of children with cancer was determined. Of special interest was the question of to what extent the incidence of bacteremias is increased by the implant. For this reason the method of matched pairs analysis was chosen comparing 55 patients with 61 catheters to 1 child each who received the therapy via peripheral veins. Apart from having the same disease, the same therapy protocol and the same age group the partners had a similar number of leukocytopenic days (leukocyte counts, < 1000/microliters) in the study period. The observation time was 9671 days in the catheter group and 9666 days in the control group. During this time 167 fever episodes (17.7 episodes/1000 days) were recorded in the patients with implant but only 133 episodes (14.0/1000 days) in the control patients. Study and control groups had similar frequencies of fever of unknown origin with leukocyte counts > or = 1000/microliters and fever with a known focus. However, 29 bacteremias (2.9 episodes/1000 days) represented a 4 times higher complication rate with the use of Broviac catheters than in the control group (7 bacteremias, 0.7 episode/1000 days). Episodes of fever of unknown origin with leukocytopenia were 1.5 times more common in the catheter group than in the control group. Although it is not possible to prove that the catheter played a role as focus of bacterial infection, an increased risk of infection must be supposed. The Broviac catheter meets with broad approval by the patients, parents and medical staff.(ABSTRACT TRUNCATED AT 250 WORDS)


Prophylaxis of ophthalmia neonatorum: comparison of silver nitrate, tetracycline, erythromycin and no prophylaxis.

From November, 1989, to October, 1991, 4544 neonates were born at our hospital. Neonatal ocular prophylaxis immediately after birth was used with 1% tetracycline ophthalmic ointment in 1156 neonates, 0.5% erythromycin ophthalmic ointment in 1163 neonates and 1% silver nitrate drops in 1082 neonates. No prophylaxis for neonatal conjunctivitis was given to 1143 neonates. A total of 302 infants (6.7%) developed conjunctivitis during the first 4 weeks of life. Between December, 1991, and January, 1992, 425 neonates were born at our hospital and all were given 0.5% erythromycin ophthalmic ointment twice in the first 24 hours after birth for ocular prophylaxis. Thirty-one (7.3%) infants developed conjunctivitis during the neonatal period. The incidence rates of neonatal chlamydial conjuctivitis in the tetracycline, erythromycin, silver nitrate, no prophylaxis and erythromycin twice groups were 1.3, 1.5, 1.7, 1.6 and 1.4%, respectively. We conclude that neonatal ocular prophylaxis with erythromycin (one or two doses) or tetracycline or silver nitrate does not significantly reduce the incidence of neonatal chlamydial conjunctivitis compared with that in those given no prophylaxis.


Coagulase-negative staphylococcal bacteremia in severely malnourished Jamaican children.

Immunosuppression increases the susceptibility to infection and changes the inflammatory response in children with severe protein-energy malnutrition. In this 5-year prospective study bacteremia was documented in 16% of 336 severely malnourished children, 2 to 34 months of age, who were hospitalized consecutively in the Tropical Metabolism Research Unit, Kingston, Jamaica. The 53 children had 60 episodes of nosocomial and community-acquired bacteremia with 69 blood isolates. Community-acquired bacteremia accounted for 72% (43 of 60) of bacteremic episodes. Thirty-five percent (24 of 69) of the strains were coagulase-negative staphylococci, 19% (13 of 69) were Staphylococcus aureus and 11% (8 of 69) were Streptococcus Group D. Seventeen episodes of coagulase-negative staphylococcal bacteremia were acquired in the community and 7 were nosocomial. These patients were more likely to have pneumonic consolidation than children with all other bacteremias combined (P < 0.02, Fisher's exact test). The bacteremia-related case fatality rate was 8% (5 of 60). Polymicrobial and Gram-negative septicemia were independent positive predictive factors for mortality when compared with single-agent and Gram-positive sepsis (P < 0.02). This 71% (49 of 69) prevalence of Gram-positive organisms suggests a change in the epidemiology from the predominant Gram-negative etiologies (76%) described in previous reports.


Intensive short course chemotherapy for treatment of Greek children with tuberculosis.

This prospective study with an 18-month posttreatment follow-up evaluated the efficacy of intensive short course chemotherapy in Greek children with pulmonary or extrapulmonary tuberculosis. Between November, 1988, and March 1991 a 2-month regimen of rifampin, 10 to 12 mg/kg/day, isoniazid, 10 to 12 mg/kg/day, and pyrazinamide, 30 to 35 mg/kg/day, followed by rifampin and isoniazid for the remaining 4 months, was administered orally to 36 children with tuberculosis. Twenty-three boys and 13 girls ages 8 months to 12 years (mean, 5 1/2 years) were enrolled in the study. The diagnostic criteria for establishing tuberculosis were tuberculin skin test reactivity, radiographic findings compatible with tuberculosis, epidemiological data and clinical and laboratory findings. Four children had extrapulmonary and 32 had pulmonary tuberculosis; 9 of the latter were asymptomatic. Among the pulmonary cases there were 2 children with pleural effusion. Clinical response to therapy was apparent within 7 to 14 days; the pleural effusions resolved in 2 to 6 weeks and the pulmonary infiltrates cleared in 2 to 6 months. Hilar adenopathy regressed within 18 months or longer. No serious problems with drug tolerance or toxicity were noted during the treatment period. Temporary hyperuricemia and transient elevation in serum transaminases were observed in 11 patients but no drug modification was required. There were no posttreatment relapses. These findings suggest that intensive short course chemotherapy for the treatment of Greek children with pulmonary or extrapulmonary tuberculosis appears to be effective, safe, of good patient compliance and comparable to the longer treatment regimens.


Diarrhea among African children born to human immunodeficiency virus 1-infected mothers: clinical, microbiologic and epidemiologic features.

Diarrhea and weight loss are common features of pediatric and adult human immunodeficiency type 1 (HIV-1) infection, particularly in developing countries. We studied prospectively episodes of diarrhea in 559 children, ages 10 to 15 months, participating in a longitudinal study of perinatal HIV-1 infection in Kinshasa, Zaire. Children with HIV-1 infection had more frequent episodes of diarrhea and were more likely to present with fever or moderate or severe dehydration and to have persistent or fatal diarrhea. Of 9 HIV-1-positive infants with diarrhea, 3 had enteroadherence factor-positive Escherichia coli, compared with 5 of 74 HIV-1-negative children with diarrhea (P = 0.04); no other pathogen was associated with HIV-1 infection. In a logistic regression model diarrhea was significantly associated with HIV-1 infection in the child, moderate or severe malnutrition and symptoms of acquired immunodeficiency syndrome in the mother. Diarrhea among children with perinatal HIV infection in Zaire is more severe than among uninfected children and is associated with malnutrition and advanced disease in the mother.


Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes.

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxyacetophenone, on the adenosine-5'-triphosphate(ATP)-sensitive K+ channel current (IK.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5-1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of IK.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K+ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as IK.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on IK.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of IK.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene.

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K+ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K+ channel blocker barium (10 mmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mumol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mumol/l), by cobalt (100 mumol/l), zinc (100 mumol/l), gadolinium (100 mumol/l), verapamil (10 mumol/l) and diltiazem (10 mumol/l), but are abolished in the presence of 100 mumol/l lanthanum, 1 mmol/l cadmium, 10 mumol/l nifedipine, 25 mumol/l SK & F 96365 and 200 mumol/l TMB-8. Stimulation of calcium entry by 10 nmol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.


Substrate specificity of the luminal Na(+)-dependent sulphate transport system in the proximal renal tubule as compared to the contraluminal sulphate exchange system.

The efflux of [35S]sulphate from the lumen of the proximal renal tubule into tubular cells of rats was measured by the stop-flow tubular-lumen microperfusion technique. The transport parameters obtained and the apparent Ki values of competing substrates were compared with those of the contraluminal influx of [35S]-sulphate from the interstitium into tubular cells. For the luminal sulphate efflux a Km(l, SO4(2-)) of 0.8 mmol/l and a Jmax(l, SO4(2-)) of 0.2 pmol s-1 cm-1 were found. The corresponding contraluminal values were Km(cl,SO4(2-)) 1.4 mmol/l and Jmax(cl,SO4(2-)) 1.2 pmol s-1 cm-1. Omission of Na+ from the perfusates reduced the luminal efflux of sulphate by 83%, while the contraluminal influx of sulphate was not changed. Increase in HCO3- concentration inhibited both luminal efflux and contraluminal influx of sulphate, while a change of pH from 6.0 to 8.0 was without effect. Comparing the apparent Ki(SO4(2-)) values for luminal and contraluminal sulphate transport, a relationship close to 1:1 was seen for some inorganic substrates with tetrahedral molecular structure (thiosulphate, sulphate, molybdate and selenate). The same holds for phosphate, while for oxalate the contraluminal Ki(SO4(2-)) value was lower than the luminal one (1.2 and 4.5 mmol/l). Some of the dicarboxylates and disulphonates tested show the same affinity to the luminal Na(+)-dependent sulphate transporter and the contraluminal sulphate exchange system, whereas most of the benzene carboxylate and benzenesulphonate derivatives tested exhibit higher luminal than contraluminal Ki values. The inhibitory potency increased with rising numbers of substituents on the benzene ring. This effect was more pronounced for the contraluminal sulphate transporter. In general, only disulphonates and analogues as well as similarly structured compounds (5-sulphosalicylate, 2-hydroxy-5-nitrobenzenesulphonate, eosine-5-isothiocyanate) have a good inhibitory potency toward the luminal sulphate transporter [apparent Ki 0.9-3.1 mmol/l]. All the tested sulphamoyl and phenoxy diuretics, and fluorescein and phenolphthalein dyes showed no or a smaller inhibitory potency to the luminal sulphate transport system than to the contraluminal. The most effective inhibitors of both sulphate transport systems are 8-anilino-1-naphthalenesulphonate, orange G, and H2-DIDS. The data indicate that the Na(+)-dependent luminal and the Na(+)-independent contraluminal sulphate transport systems accommodate a similar spectrum of anionic substrates, whereby the inhibitory potency against the luminal Na(+)-dependent sulphate transport system is identical or smaller than against the contraluminal transporter.


Lack of effect of intraluminal pressure on renin release from isolated afferent arterioles.

To evaluate the role of the proposed baroreceptor mechanism in the afferent arteriole in regulating renin release, we modified the isolated perfused tubule technique to perfuse afferent arterioles. Arterioles with attached glomeruli were isolated from rabbit kidneys and perfused using standard methods. To stop the arteriolar flow and allow perfusion pressure, as set by a mercury manometer, to be built up in the lumen of the vessel, the glomerulus was sucked into a constriction pipette. The preparation was continuously superfused with Krebs-Ringer solution in the first series of experiment, and a cell culture medium in the second series of experiment. The superfusate droplets were collected under mineral oil with 10-min collection intervals. The renin content of the samples was assayed by radioimmunoassay of the angiotensin I generated. In the two series of experiments we tested the effects of sequential changes in intraluminal pressure on renin release. In the first series of experiments (n = 6) the renin release was 56.3 nGU arteriole-1 min-1 in the first 10 min of sampling. The renin release was then constant for 80 min with an average of 21.6 nGU arteriole-1 min-1. In the last 30 min the renin release was 96.5 nGU arteriole-1 min-1. In the second series of experiments (n = 8) the renin release was 26.5 nGU arteriole-1 min-1 throughout the course of the experiment. These results indicate that under these conditions there is no relation between renin release and intraluminal pressure in afferent arterioles.


Relation between slow-wave frequency and spiking activity during the migrating myoelectric complex in dogs.

The quantitative relation between slow-wave periods and spiking activity was evaluated in vivo in canine small intestine during the fasted state. Experiments were performed in three conscious dogs with three bipolar electrodes, implanted respectively 10, 25 and 40 cm beyond the ligament of Treitz. Digitized electrical recordings were automatically processed for the individual slow-wave periods and spike-burst intensities using a set of computer programs developed in our laboratory. A linear correlation existed between the degree of spiking activity and the average length of the preceding slow-wave period. The slopes of the regression lines were less steep for more distal electrodes. A second series of experiments showed that an increase in the slow-wave period precedes the onset of phase 3 of the migrating myoelectric complex and that a fall in slow-wave period precedes the end of phase 3. These data show that a low slow-wave frequency is accompanied by a facilitation of spiking activity, whereas shortening of the slow-wave period is accompanied by a decrease in spike burst intensity. This relation between slow-wave period and spiking activity shows an aboral trend that may be related to intrinsic slow-wave frequency.


Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes.

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239-241], the effect of EGF on 45Ca2+ uptake and on 22Na+ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca2+, whereas Na+ and K+ were varied. In 140 mM Na+/5 mM K+ medium EGF (250 ng/ml) stimulated 45Ca2+ uptake by 50%-90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K+ up to 75 mM at the expense of extracellular Na2+ stimulated the EGF-induced 45Ca2+ uptake by about twofold compared to 145 mM Na+ medium both in quin-2-loaded and in untreated cells. In 145 mM K+ medium, however, no EGF-induced 45Ca2+ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca2+ entry was stimulated twofold by EGF. After increasing intracellular Na+ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K+ medium, 45Ca2+ uptake induced by EGF gradually increased. In contrast, in 140 mM Na+/5 mM K+ as well as in 70 mM Na+/75 mM K+ medium, 45Ca2+ uptake accelerated by EGF was largely unaffected by a modified red cell Na+ content. When 22Na-loaded untreated red cells were suspended in 145 mM K+ medium EGF stimulated red cell 22Na+ efflux by more than threefold. In 140 mM Na+/5 mM K+ as well as in 70 mM Na+/75 mM K+ medium, no 22Na+ efflux induced by the growth factor was evident.(ABSTRACT TRUNCATED AT 250 WORDS)


Involvement of a pertussis toxin-sensitive G-protein in excitation-contraction coupling of intact and cut-end voltage-clamped skeletal muscle fibres.

In voltage-clamped frog muscle fibres 10 ng/ml PTX induced a decrease (approximately 35%) of tension when applied externally. Internal application in cut-end fibres significantly depressed tension after 20 min. This effect increased with time to reach 65% after 60 min. PTX shifted the voltage-dependent inactivation curve of tension by 30 mV towards hyperpolarizations and this was counteracted by raising external calcium concentration. The toxin induced a parallel decrease in tension and voltage-sensitive charge movement (49 +/- 9% and 52 +/- 6% respectively; n = 6). This was not counteracted by prior impregnation with forskolin. Internally applied GTP gamma S (500 microM) induced a simultaneous increase in tension (57 +/- 5%) and charge amount displaced (40 +/- 7%). By contrast, GDP beta S decreased tension and charge movement by 35 +/- 5% and 36 +/- 6% respectively.


Characterization of the self-splicing products of a mobile intron from the nuclear rDNA of Physarum polycephalum.

We have characterized the splicing products formed in vitro from RNA derived from the mobile group I intron in the nuclear rDNA of Physarum polycephalum, Pp LSU 3. This intron is a close relative of the well known Tetrahymena intron Tt LSU 1, being inserted at exactly the same position in the rDNA and sharing about 90% sequence identity with Tt LSU 1 in the conserved elements characteristic of the catalytic core of all group I introns. However, Pp LSU 3 differs from Tt LSU 1 in that it encodes a site-specific endonuclease, which mediates the homing of the intron to unoccupied target sites. The endonuclease, I-Ppo, would appear to be a unique example of a protein encoded by an RNA polymerase I transcript. To gain clues to the splicing products formed in vivo, and to the nature of the messenger RNA for I-Ppo, we subjected Pp LSU 3 RNA to standard self-splicing conditions in vitro, and then analyzed the products by size, by northern blotting, and by primer extension. The results show two novel features. First, in addition to the expected 5' splice site, there is an alternative 5' splice site in the upstream exon, just preceding the first codon of the I-Ppo open reading frame. Second, at the position corresponding to the major circularization site in Tt LSU 1 there is an internal processing site, leading to the efficient separation of two halves of the excised intron, the 5' half encoding I-Ppo and 3' half containing the ribozyme. Surprisingly, this cleavage appears not to be due to circularization followed by hydrolytic opening of the circle, but rather to G addition. The formation of these products in vitro suggests how the messenger RNA for the I-Ppo endonuclease may be generated in vivo.


Cofractionation of the TATA-binding protein with the RNA polymerase III transcription factor TFIIIB.

We have investigated the requirement for TBP (TATA-binding protein) in transcription mediated by RNA polymerase III (pol III) in fractionated HeLa cell extracts. Two activities, TFIIIB and TFIIIC, found in phosphocellulose fractions PC B and PC C respectively, have been defined as necessary and sufficient, with pol III, for in vitro transcription of tRNA genes. Depletion of TBP from PC B, using antibodies raised against human TBP, is shown to inhibit the pol III transcriptional activity of the fraction. Furthermore, TBP is present in fractions with human TFIIIB activity, and a proportion of TBP cofractionates with TFIIIB over four chromatographic purification steps. TFIIIB fractions are capable of supplying TBP in the form necessary for pol III transcription, and cannot be substituted by fractions containing other TBP complexes or TBP alone. The use of a 5S RNA gene and two tRNA templates supports the general relevance of our findings for pol III gene transcription. Purified TFIIIB activity can also support pol II-mediated transcription, and is found in a complex of approximately 230kD, suggesting that TFIIIB may be the same as the previously characterized B-TFIID complex (1,2). We suggest that transcription by the three RNA polymerases is mediated by distinct TBP-TAF complexes: SL1 and D-TFIID for pol I and pol II respectively, and TFIIIB for pol III.


Elongation factor-1 messenger-RNA levels in cultured cells are high compared to tissue and are not drastically affected further by oncogenic transformation.

Copy-DNA clones covering the complete coding sequence of human Elongation Factor-1 gamma mRNA have been isolated and characterized. The expression of Elongation Factor-1 in a variety of cell lines and a number of tissues shows a large increase in Elongation Factor-1 mRNA going from tissue to cultured cells (20-fold). Messenger-RNA levels for Elongation Factor-1 alpha, -1 beta and -1 gamma increase in parallel suggesting coordinate regulation of the expression of these genes. Oncogenic transformation in vitro does not strongly affect Elongation Factor-1 mRNA levels.


Pseudouridine in the anticodon G psi A of plant cytoplasmic tRNA(Tyr) is required for UAG and UAA suppression in the TMV-specific context.

We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.


The FLP protein contacts both major and minor grooves of its recognition target sequence.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.


Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA.

To determine the sequence requirements and structural features of the self-cleavage domain of hepatitis delta virus (HDV) antigenomic RNA, we constructed a series of mutants and measured the rate constant of the cleavage reaction for each. The self-cleavage activity of HDV RNA of antigenomic sense was found to reside in a region of less than 90 nucleotides in length. The catalytic domain contained a long complementary sequence which could be deleted to half of its original size. Moreover, this region could be replaced by other sequences as long as they could fold into a stem-and-loop structure. The catalytic domain also required a 6-basepair helix adjacent to the cleaving point for activity. The structural features of these two base-pairing regions are quite similar to those of the HDV genomic self-cleavage domain. The cleavage site as well as the the hinge region (the sequence between the two stems) requires specific sequences for activity.


Detection and characterization of a ring chromosome in the fission yeast Schizosaccharomyces pombe.

NotI and SfiI genomic restriction maps were used to detect and characterize a ring chromosome II in a Schizosaccharomyces pombe strain with a meiotic defect on chromosome II. The ring chromosome was formed by an intrachromosomal fusion near, or at, the very ends of chromosome II.


Retinoblastoma-repression of E2F-dependent transcription depends on the ability of the retinoblastoma protein to interact with E2F and is abrogated by the adenovirus E1A oncoprotein.

The product of the retinoblastoma tumor suppressor gene interacts with the transcription factor E2F. Two distinct types of interactions can be detected between the retinoblastoma gene product (Rb) and E2F. The first type involves an Rb-binding protein, RBP60. The Rb/E2F complex formed in the presence of RBP60 is able to bind DNA and migrates with a distinct mobility in gel retardation assays. The second type of Rb/E2F complex is seen in the absence of RBP60. This second type of Rb/E2F complex does not form a band-shift complex in gel retardation assays and its formation results in an apparent inhibition or loss of the DNA binding activity of E2F. Using a series of Rb-mutants we show that these two types of Rb/E2F complexes depend on common domains of the Rb protein. The T/E1A-binding region as well as the carboxyl-terminus of the Rb protein are critical for these two types of Rb/E2F interactions. We also show that the retinoblastoma protein represses the E2F-dependent transcription, and this Rb-repression of the E2F-dependent transcription depends on the ability of Rb to interact with E2F. Moreover, the adenovirus E1A gene product, which binds Rb, counteracts the Rb-repression and restores E2F-dependent transcription.


Regulatory gene INO4 of yeast phospholipid biosynthesis is positively autoregulated and functions as a transactivator of fatty acid synthase genes FAS1 and FAS2 from Saccharomyces cerevisiae.

The sequence motif 5' TYTTCACATGY 3' functions as an upstream activation site common to both yeast fatty acid synthase genes, FAS1 and FAS2. In addition, this UASFAS element is shared by all so far characterized genes of yeast phospholipid biosynthesis. We have investigated the influence of a functional INO4 gene previously described as a regulator of inositol biosynthesis on the expression of FAS1 and FAS2. In a delta ino4 null allele strain, both genes are expressed at only 50% of wild type level. Using individual UASFAS sequence motifs inserted into a heterologous test system, a drastic decrease of reporter gene expression to 2-10% of the wild type reference was observed in the delta ino4 mutant. In gel retardation assays, the protein-DNA complex involving the previously described FAS binding factor 1, Fbf1, was absent when using a protein extract from the delta ino4 mutant. On the other hand, this signal was enhanced with an extract from cells grown under conditions of inositol/choline derepression. Subsequent experiments demonstrated that INO4 expression is itself affected by phospholipid precursors, mediated by an UASFAS element in the INO4 upstream region. Thus, in addition of being an activator of phospholipid biosynthetic genes, INO4 is also subject to a positive autoregulatory loop in its own biosynthesis.