Reduction of target gene expression by a modified U1 snRNA.

Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.

Immunocytochemical localization of enkephalin in the neostriatum of rat brain: a light and electron microscopic study.

The peroxidase-antiperoxidase (PAP) immunocytochemical technique was used to determine the light and electron microscopic localization of antisera directed against either methionine [Met5]-or leucine [Leu5]-enkephalin in the neostriatum of brains from untreated rats. By light microscopy, neuronal perikarya and processes showing enkephalin-like immunoreactivity (ELI) were unevenly distributed throughout the neostriatum. The greatest accumulation of neuronal structures showing ELI was in the ventro- and caudo- lateral portions of the nucleus. The labeled perikarya measured 10--15 micrometer in diameter and constituted about 15--20% of the total neurons in the neostriatum. By electron microscopy, examination of three areas from horizontal and coronal sections revealed no regional differences in types of neurons showing ELI or in their synaptic organization. All labeled neurons showed a relatively low intensity reaction product which was diffusely distributed throughout the perikarya and dendrites. The cytoplasm contained relatively few organelles, which included mitochondria, endoplasmic reticulum and numerous "alveolate" vesicles. The dendrites had many spiny processes which formed asymmetric synapses with unlabeled axon terminals containing all small clear vesicles. In contrast to the perikarya and dendrites a dense accumulation of reaction product was present in a few myelinated and numerous unmyelinated axons and axonal varicosities. Approximately 75% of the labeled varicosities did not form a specialized synaptic junction in a single plane of section. The remaining 25% of the labeled terminals formed asymmetric junctions primarily with unlabeled dendrites and rarely with unlabeled perikarya or axons. The morphology and synaptic relations of the neurons showing ELI suggest that they may belong to the general group of medium-sized spiny cells characterized in Golgi studies by Kemp and Powell ('71a). At least some of the peptide-containing neurons may also have a myelinated efferent axons.

Treatment of estrogen-resistant stage D carcinoma of prostate with cis diamminedichloroplatinum.

Forty-five patients with rapidly progressive, estrogen-resistant Stage D adenocarcinoma of the prostate were treated with infusions of cis diamminedichloroplatinum (cis platinum) (1 mg./Kg./week) for six weeks initially and every three weeks thereafter. A partial objective response was observed in 13 of 45 patients (29 per cent). This response lasted from two to sixteen months with an average of six months. Eighteen patients (40 per cent) had a significant decrease or disappearance of bone pain and became ambulatory. Six patients (13 per cent) remained stable, and 8 patients (18 per cent) did not respond to treatment and showed progression of their disease. The toxicity of the treatment was mild to moderate. Most of the patients were treated in the outpatient department. Cis platinum appears to be the most effective drug available to date for the treatment of advanced carcinoma of the prostate.

Chemotherapy in metastatic, hormone refractory prostatic cancer using chlorambucil in combination with prednisolone versus conjugate, prednimustine (Leo 1031).

Chlorambucil plus prednisolone were administered to 11 patients with metastasis hormone refractory prostatic cancer, and the results were contrasted with a previously reported series of 23 similar patients treated with the chemical conjugate of these two agents, known as prednimustine or Leo 1031. The conjugated form of treatment (Leo 1031) had a limited therapeutic advantage, in that 3 patients experienced shrinkage of an enlarged prostate, 2 of whom also had elevated acid phosphatase levels return to normal and 5 others experienced only subjective improvement. There were, however, more adverse side effects in this group than those noted in patients treated with the combination of agents. Patients treated with the combination of drugs experienced no appreciable tumor shrinkage and none had acid phosphatase return to normal, although some reduction was noted in 8 of 11 patients who had elevated levels initially. Two of the 11 patients were considered stable for twelve months and one other remained ambulatory with mild pain for six months. Thus, to the degree that these studies permit, it is judged that the conjugated agent may have some limited therapeutic advantage not observed when the unconjugated agents were used.

Estramustine phosphate with multiple cytotoxic agents in treatment of advanced prostatic cancer.

Twenty-five patients with histologically proved adenocarcinoma of the prostate were divided into two groups and submitted to combination therapy with estramustine (Estracyt), cyclophosphamide (Cytoxan), 5-fluorouracil, and Cisplatin. Group A consisted of 10 patients newly diagnosed Stage D disease with no prior treatment. Group B consisted of 15 Stage D patients who had become hormonally unresponsive. Group A patients demonstrated an initial 100 per cent response rate including 70 per cent partial objective responses and 30 per cent stabilizations. Group B patients had a 46 per cent response with 39 per cent complete and partial responses and 6 per cent as stabilized. Toxicity was tolerable judged by the NPCP criteria. Both groups of patients are still under study for up to two years to determine if this therapy is superior to other traditional therapies.

Fine-needle aspiration of metastatic lesions and regional lymph nodes in genitourinary cancer.

A percutaneous fine-needle aspiration biopsy of metastatic tumor and/or retroperitoneal, pelvic, or abdominal lymph nodes was accomplished in 101 patients. A diagnosis of metastatic disease was confirmed by this method in 49 patients. In 26 patients, surgical excisional biopsy or exploratory laparotomy was avoided. An unknown pelvic mass causation was diagnosed in 3 patients. In 15 of 57 patients with localized bladder, prostate, or testis cancer, involvement of regional lymph nodes was confirmed by transabdominal find-needle aspiration. All 57 transabdominal aspirations were performed without complications. The method is inexpensive, safe, and can have a high accuracy in diagnosing a local tumor spread or distant metastases.

High-dose tamoxifen in metastatic renal cell carcinoma.

The effectiveness of high-dose Tamoxifen in the treatment of metastatic renal cell carcinoma was evaluated. Fifteen patients were treated for eight weeks with 80 mg of Tamoxifen daily. Side effect were minimal. Results showed progression of disease in 47 per cent of the patients, no change in disease was seen in 40 per cent, and 13 per cent showed a partial objective response. Twenty per cent of patients showed deterioration of performance status. High-dose Tamoxifen does not appear to be useful in the treatment of advanced renal cell carcinoma.

Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum.

The morphology and synaptic associations of dopaminergic axons in the n. caudate-putamen (neostriatum) of the adult rat brain are examined. Identification of dopaminergic axons is based upon the electron microscopic immunocytochemical localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase. Immunoreactivity for the enzyme is detected in unmyelinated axons and axon terminals in serial sections collected throughout the neostriatum. The labeled terminals range from 0.1 to 1.5 micron in diameter and have peroxidase reaction product located around closely packed, round vesicles with a diameter of 40-60 nm. The tyrosine hydroxylase containing axon terminals constitute approximately 21% of the total number of terminals in the n. caudatus-putamen and include 3 types which differ in size and synaptic specializations. The most prevalent (82% of total), type I, is small (0.15-0.39 micron in diameter) and forms symmetric junctions with dendrites and dendritic spines. The other two terminal types (II and III) have a medium to large diameter (0.4-1.5 micron) and show either no membrane specializations or asymmetric junctions with dendrites. The axon terminals without observable membrane densities are occasionally oriented so as to suggest an association with dopaminergic and non-dopaminergic axon terminals. These findings indicate that while the dopaminergic terminals may form axoaxonic connections, the primary synaptic contacts are with dendrites of intrinsic neurons in all regions of n. caudatus-putamen.

Umbilical cord blood gas analysis at the time of delivery.

AIMS: it is now recommended that cord blood acid-base measurement is performed routinely at time of delivery in the UK as a measure of fetal response to labour. However, there remains some uncertainty about the value of this procedure. In this paper our experience of cord blood analysis is described and the literature is reviewed to: (1) provide an overview of the physiological basis of cord blood acid-base assessment; (2) describe the appropriate methodology and identify issues which have contributed to confusion and undermined the value of cord blood sampling; and (3) address the practical issues of cord blood sampling. CONCLUSIONS: cord blood acid-base measurement has a sound physiological basis. It provides objective information which is a useful adjunct to subjective methods of newborn assessment, enables babies at risk of neonatal morbidity to be identified, can be helpful in litigation cases and is a prerequisite for clinical audit. However, to be of benefit the information must be correct and correctly interpreted.

The QSHP proposal: 10 questions most often asked.

Qualified Student Health Plans (QSHPs) have gained increasing support since the authors introduced the proposal to the field of college health in 1994. This article answers 10 of the most frequently asked questions about QSHPs and summarizes changes in the proposal that have resulted from discussions among college health professionals throughout the country. The QSHP proposal, which has been endorsed by the board of directors of the American College Health Association, offers the following benefits for the college health field and for students: It is founded on the traditional prepaid college health model and assures that control remains with higher education; participation is voluntary for employers and for institutions of higher education; no new tax funding is required; QSHPs preclude state interference and provide employers with incentives to contribute to the cost of student healthcare; both the cost of student health insurance and duplication of coverage are reduced and benefits are shifted to where the students live; the problem of uninsured and underinsured students is eliminated at participating institutions. With incremental healthcare reform continuing at the state level, the time to move forward with the proposal is viewed as ideal.

Student healthcare delivery and financing programs: adapting to healthcare reform.

College health professionals want to assure the unique healthcare and health education needs of college students will continue to be met under national and state healthcare reform. This may be an "all or nothing" proposition. Either colleges and universities will have exclusive control of healthcare delivery for the college student population or else college health will not be a major force in healthcare reform. If college health is to play a meaningful role in future government-controlled health insurance programs, it must first demonstrate that current health services and insurance financing programs meet minimum quality standards. This proposal calls for expanding existing federal laws to create qualified student health plans and integrating the college health model into a reform package based on employer-sponsored health insurance. The concept of qualified student health plans allows for a high degree of flexibility that can be integrated into the majority of state and federal healthcare reform proposals, including the plan proposed by President Clinton, that are not based on a single-payer system. Ultimately, the authors suggest, their proposed plan would eliminate the current situation, in which large numbers of college students are uninsured or underinsured.

The chemotherapy of prostatic carcinoma.

Results of the first randomized trial of the National Prostatic Cancer Project (NPCP) revealed an advantage for cytoxan and 5-FU over standard therapy in hormonally resistant stage D prostate carcinoma. A subsequent trial for patients previously irradiated, receiving the less myelosuppressive agents estracyt or streptozotocin also revealed an advantage over standard therapy. Other completed randomized trials have revealed activity for prednimustine and DTIC. Trials underway for newly diagnosed stage D disease or for stage D disease clinically stable to diethylstilbestrol (DES) show promising activity for cytoxan and DES combined. Current randomized trials in advanced disease are comparing methyl CCNU and hydroxyurea wih cytoxan, and estracyt or vincristine alone or in combination. Chemotherapy in earlier staged patients as adjuvants to definitive surgery or irradiation is underway in two trials, comparing the effects of cytoxan or estracyt as long-term therapies with no additional treatment.

Modern concepts of acid and alkaline phosphatase measurement.

Three new assay methods for prostatic acid phosphatase are described relative to results of biochemical methods. Methods for total and isozymes of alkaline phosphatase are also described. Results of a field trial for acid phosphatase tests among multiple institutions in the United States revealed a marked increase in sensitivity for one assay (CIEP) over biochemical methods. which was positive with increasing frequency as the clinical and surgical stage advanced beyond B. Only for stage D did the biochemical method approach the sensitivity of the CIEP assay. The CIEP assay has potential value as a screen for prostate cancer. Another acid phosphatase assay (RIA) proved difficult to perform for many institutions in the field trial. A new solid phase immunofluorescent assay recently introduced may on further testing be more sensitive than the CIEP assay. Total alkaline phosphatase levels studied in patients with advanced disease were markedly elevated in over one-third of the cases. Higher levels were associated with poor survival or response to chemotherapy. Alkaline phosphatase isozyme levels were of added value in locating the site of the increased alkaline phosphatase activity in soft tissue, bone, or liver. Occasionally in the face of a normal total value, increased isozyme activity in one of the aforementioned compartments preceded clinical ability to detect the metastatic foci.

Adjuvant chemotherapy for bladder cancer with doxorubicin hydrochloride and cyclophosphamide: preliminary report.

We describe 25 patients with bladder cancer who received adjuvant chemotherapy with doxorubicin hydrochloride and cyclophosphamide after radical cystectomy. Two patients had stage A disease, 3 had stage B, 3 had stage C and 17 had stage D. The 2 patients with stage A tumors have been free of disease for 12 and 15 months, respectively, and the 3 patients with stage B tumors have been free of disease for an average of 25 months. Of the 3 patients with stage C tumors 2 have been free of disease for an average of 34.5 months. Of the 17 patients with stage D tumors 10 have been free of disease for an average of 1 year (59 per cent). These preliminary results seem to indicate the value of adjuvant chemotherapy with doxorubicin hydrochloride and cyclophosphamide in cases of bladder cancer.

The development and evaluation of a computer-assisted teaching programme for intrapartum fetal monitoring.

OBJECTIVE: The development and evaluation of a computer-assisted teaching programme of cardiotocography and acid-base balance. DESIGN: Randomised controlled trial. PARTICIPANTS: One hundred and seventeen midwifery and obstetric staff at Derriford Hospital, Plymouth. METHODS: The obstetricians and midwives were randomly allocated to use the teaching programme, either early or late. The late group (control) used the teaching programme three months after the early group. To assess the effect of the teaching programme, participants were tested on four occasions over eight months by a multiple choice questionnaire. Two questionnaires on ease of use were also completed. MAIN OUTCOME MEASURES: Multiple choice questionnaire scores and opinion questionnaire results. RESULTS: The mean score in the early group improved from 50-8% (test 1, pre-teaching programme) to 70.2% (test 2, post-teaching programme). The mean score in the control group was 50.3% (test 1) and 54.8% (test 2). Knowledge was retained up to seven months. CONCLUSIONS: The teaching programme was effective in improving knowledge of acid-base balance and cardiotocography and can be used by all staff whilst on duty on the labour ward.

Delegation to assistive personnel by school nurses--one state's experience.

Budget cuts to education, reduction of school health services and an increase in the nursing needs of school-age children have placed pressure on school nurses to delegate nursing procedures to assistive personnel. This paper reports on a study of school nurses in a Midwestern state. Nurses reported refusing to delegate, delegating against their better judgment, delegating without knowledge of the assistant's qualifications or without providing orientation or training, and delegating because they were told to by someone else. The study identified factors considered by nurses when deciding to delegate a procedure, educational preparation for delegation, and problems experienced with delegation.