Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides.

The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.

Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations.

Quantitative capillary gel electrophoresis (QCGE) has been developed for the accurate quantitation of a 21-mer phosphorothioate oligonucleotide, ISIS 2922, and its degradation products in an intravitreal formulation. The electrokinetic mode of injection employed by CGE necessitates formulation of the external reference standard in a sample matrix similar to that of the drug product and the use of an internal standard for improved accuracy and precision. The analytical method detailed in this paper has demonstrated the necessary accuracy, precision, linearity, range, selectivity and ruggedness for use in routine drug product analysis and stability monitoring of phosphorothioate oligonucleotides.

An international foodborne outbreak of shigellosis associated with a commercial airline.

OBJECTIVE: To determine the source of an international outbreak of shigellosis associated with consumption of food served by a Minnesota-based airline. DESIGN: Cohort studies of players and staff of a Minnesota-based professional football team and passengers on flights with a confirmed case of outbreak-associated Shigella sonnei infection. SETTING: Community- and industry-based studies conducted from October through November 1988. PARTICIPANTS: Sixty-five football team players and staff, and 725 airline passengers in the cohort studies. RESULTS: Twenty-one (32%) of 65 football players and staff developed shigellosis that was associated with consumption of cold sandwiches prepared at the airline flight kitchen (relative risk [RR], 17.1; 95% confidence interval [Cl], 2.4 to 120; P < .001). Confirmed or probable shigellosis was identified among 240 passengers on 219 flights to 24 states, the District of Columbia, and four countries between September 14 and October 13. An outbreak-associated strain of S sonnei was isolated from football players and staff, airline passengers, and flight attendants. Thirty (4.1%) of 725 passengers on 13 flights with confirmed cases had confirmed or probable shigellosis. Illness was associated with consumption of cold food items served on the flights and prepared by hand at the airline flight kitchen (RR, 5.7; 95% Cl, 1.4 to 23.5; P < .01). CONCLUSIONS: This international outbreak of shigellosis was identified only because of the occurrence of an index outbreak involving a professional football team. Prevention of Shigella transmission in mass catering establishments may require reduction of hand contact in the preparation of cold food items or elimination of these items from menus.

HRSA's role in primary care and public health in the 1990s.

The principal responsibility of the Health Resources and Services Administration (HRSA) is to see to it that primary health care services and qualified health personnel and facilities are available to all Americans, particularly the disadvantaged and the underserved. HRSA's resources are directed toward not only the most financially, functionally, and culturally vulnerable segments of the population but also to those who have significant clinical needs such as pregnant women, children, those infected with HIV. The Agency seeks to carry out its mission in many ways. The central approach, however, is to assure the availability of services to its constituencies directly or indirectly through the more than 50 programs it administers. This article explains HRSA's role in detail and cites its many ramifications for the nation's health in the 1990s.

[Revitalization of the federal and state efforts to promote community-based primary care systems]. / Revitalización de los esfuerzos federales y estatales para promover sistemas de atención primaria basados en la comunidad.

Progress toward a strengthened public health system is clearly emerging in the United States, but still faces a number of financial and functional barriers. There is no question, however, that the importance being placed on primary care systems is making a difference as more and more communities are seeking and implementing effective approaches that bring resources together in ways that work. That, of course, is the ultimate objective; that is, that all public agencies assure their constituents that their needed services are being provided in an affordable way.

The determination of recombinant human tissue-type plasminogen activator activity by turbidimetry using a microcentrifugal analyzer.

A method is described for determining the activity of recombinant human tissue-type plasminogen activator (rt-PA) by turbidimetry using a microcentrifugal analyzer (MCA). A mixture of thrombin and rt-PA is centrifuged into a mixture of fibrinogen and plasminogen to initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance versus time is analyzed to determine the assay endpoint. Different rt-PA assay concentration ranges were studied in conjunction with profile endpoints for assay optimization. Spiked placebo recovery studies were used to evaluate assay accuracy and precision, which were determined to be 99.5 and 5% (relative standard deviation or RSD), respectively. Assay sensitivity was 0.5 ng/ml. Typical analysis time, including calculations, for a standard curve plus 14 samples was less than 30 min. The application of turbidimetry with the MCA for determining rt-PA activity provides rapid sample analysis and high throughput while maintaining accuracy and precision.

Effects of health locus of control and health value upon EMG biofeedback training.

Subjects who scored highly internal or external on a health locus of control scale and who scored high or low on the value each attached to good health were each given two sessions of frontalis EMG biofeedback training in accordance with a 2 x 2 x 2 factorial design. It was predicted that both internality and high health value would be positively associated with ability to control EMG and that these influences would combine additively. The predictions were not supported but a significant interaction between internality and health value was found. This interaction could be explained by assuming that both internality and high health value increased drive which in turn produced greater muscle tension, the relationship between drive and muscle tension being an ascending sigmoid function.

Pseudogynecomastia secondary to injection of heroin into breast tissue.

A 50-year-old man who has a heroin addict developed bilateral, symmetrical swelling of the breasts as a result of injecting himself directly into the breasts for several years. Results of histologic examination of the breast tissue showed granulomatous inflammation and a foreign body reaction without gynecomastia or tumor. Liver and endocrine functions were generally normal.

Effects of prior experience and 'functional disturbance' on acute and chronic tolerance to methylphenidate.

The degree of habituation or adaptation an animal has experienced prior to the administration of methylphenidate was found to be more crucial in determining the animal's response to the drug than chronic drug administration alone. Rats allowed to adapt in a Y-maze for 24 days showed less methylphenidate-induced activity than animals receiving chronic administration of saline or methylphenidate but no adaptation, when animals were injected with the drug and tested in the Y-maze on Day 25. Animals also were shown to develop tolerance to the methylphenidate-induced activity.

The effects of ethanol upon threshold and response rate for self-stimulation.

The hypothesis that ethanol would reduce the threshold for self-stimulation of the medial forebrain bundle was not supported. Ten rats, implanted with electrodes in the lateral hypothalamus, were shaped to bar press for electrical brain stimulation. The effects of 0.6, 0.9, and 1.2 g/kg ethanol injections upon threshold and response rate for self-stimulation were measured. The lowest dose had no effect upon self-stimulation threshold while 0.9 and 1.2 g/kg ethanol raised thresholds. Rate of bar pressing was increased by 0.6 g/kg ethanol but was not affected by higher doses. Results were discussed in terms of a postulated dual effect of ethanol upon a brain arousal system and upon a reward system.

The role of locomotion in conditioning methylphenidate-induced locomotor activity.

This experiment determined whether overt performance of the entire response (actual running) was necessary for the conditioning of methylphenidate-induced locomotor activity (wheel-running) in guinea pigs. Four guinea pigs were given daily injections of 2.5 mg/kg methylphenidate and were allowed to run in activity wheels; 4 other guinea pigs were given methylphenidate and were placed in locked activity wheels; a third group of 4 guinea pigs were administered saline and allowed to locomote; a fourth group of 4 guinea pigs received saline injections and were placed in locked activity wheels. After 12 days of injection, all animals were given saline injections on the 9 subsequent days and allowed to run freely in the wheels. The 2 groups which had received methylphenidate showed more locomotor activity than the saline injected animals but were not distinguishable from each other on the basis of prior opportunity to engage in locomotor activity. These results were interpreted to indicate that (a) increased methylphenidate-induced locomotor activity may be conditioned with repeated administration of the drug, and (b) actual running is not essential for the conditioning of drug-induced wheel-running.

Tolerance in methylphenidate-induced locomotion in prairie dogs (Cynomys ludovicianus).

After a five-day adaptation period in activity wheels, four prairie dogs were assigned to a methylphenidate group (2.5 mg/kg, s.c.) and four prairie dogs were assigned to a saline group for ten consecutive daily injections. Half-revolutions of locomotor activity were recorded during seven measurement periods taken daily. During the third measurement period, from 1-3 hrs following injection, the methylphenidate group declined in locomotor activity over the first three days of drug administration.

Antiviral activity of arabinosyladenine and arabinosylhypoxanthine in herpes simplex virus-infected KB cells: selective inhibition of viral deoxyribonucleic acid synthesis in synchronized suspension cultures.

The drug 9-beta-d-arabinofuranosyladenine (ara-A) significantly suppressed the formation of herpes simplex virus type 1-induced syncytia in BHK-21/4 cells at concentrations as low as 0.1 mug/ml. Optimal activity was noted when the drug was added before initiation of viral deoxyribonucleic acid (DNA) synthesis (3.5 h postinfection). The deaminated derivative of ara-A, 9-beta-d-arabinofuranosylhypoxanthine (ara-H), was at least 10 times less effective in suppressing the development of herpes simplex virus-induced syncytia. The replication of herpes simplex virus was measured by assaying fluids and cells from infected drug-treated cultures by using a plaque production technique. Ara-A at drug levels of >10 < 32 mug/ml completely blocked the replication of infectious virus particles. Ara-H was less effective than ara-A in reducing the replication of virions. Rates of host and viral DNA synthesis were monitored by pulse labeling herpes simplex virus-infected synchronized KB cells with [(3)H]thymidine and subsequently separating viral from cellular DNA in CsCl density gradients. During synthetic (S) phase, ara-A or ara-H at concentrations ranging from 3.2 to 32 mug/ml selectively inhibited viral DNA synthesis. At 3.2 mug of ara-A per ml, viral DNA synthesis was reduced 74% although total cellular DNA synthesis was unaffected. Increasing concentrations of ara-A produced increasing temporal delays in the maximal rate of host DNA synthesis. This time shift was not observed in cells treated with ara-H.

Successive spatial discrimination reversals in the prairie dog.

Six prairie dogs (Cynomys ludovicianus) were given reversal training on a spatial discrimination reversal task in a Wisconsin General Test Apparatus (WGTA). As with many other species, more errors occurred on the first reversal problem than during acquisition of the original discrimination. However, performance improved over training until each S eventually reversed after a single nonreinforced error. Initial findings indicate that prairie dogs perform at high levels in discrimination learning experiments and could be valuable subjects for psychological research.

Attenuation of "acute" habituation by scopolamine in the black-tailed prairie dog (Cynomys ludovicianus).

The hypothesis after scopolamine attenuates habituation occurring within a training session as well as that occurring between training sessions was tested and confirmed. Scopolamine injections (.5 mg/kg) reduced spontaneous wheel running in the prairie dog. The same dosage did not affect the threshold for wheen running induced by electrical brain stimulation (EBS). Another experiment demonstrated that EBS-induced running declined during a 10-min. stimulation period and indicated that the decline was habituative since the response was repeatedly dishabituated. In a final experiment .5 mg/kg of scopolamine reduced habituation of EBS-induced wheel running during 10-min. sessions.