Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins.

Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.

Development of rebiana, a natural, non-caloric sweetener.

Rebiana is the common name for high-purity rebaudioside A, a natural non-calorie sweetener 200-300 times more potent than sucrose. It provides zero calories and has a clean, sweet taste with no significant undesirable taste characteristics. It is functional in a wide array of beverages and foods and can be blended with other non-calorie or carbohydrate sweeteners. It is stable under dry conditions, and has much better stability than aspartame or neotame in aqueous food systems. Studies undertaken for the development of a purification process and for the full characterization of the properties of rebiana are reported here.

Inhaled endothelin A antagonist improves arterial oxygenation in experimental acute lung injury.

OBJECTIVES: To study the effects of an inhaled endothelin A (ET(A)) receptor antagonist on hemodynamics and pulmonary gas exchange in experimental acute lung injury (ALI). DESIGN AND SETTING: Prospective, randomized, and controlled study in a university laboratory. PARTICIPANTS AND INTERVENTIONS: Sixteen pigs were ventilated in a volume controlled mode during general anesthesia. ALI was induced by surfactant depletion using repetitive lung lavages until the PaO2/FIO2 ratio was below 100 mmHg. The animals were then randomly assigned to receive either a nebulized ET(A) receptor antagonist (LU-135252, 3 mg/kg, inhaled over 1 h; LU group) or nebulization of saline (5-10 ml inhaled over 1 h) with no further intervention (controls). MEASUREMENTS AND RESULTS: Parameters of hemodynamics and gas exchange were measured for 6 h after induction of ALI. In the LU group intrapulmonary right-left shunting (QS/QT) decreased from 58 +/- 8% at the onset of ALI to 27 +/- 12% 3 h and 24 +/- 9% 6 h after ALI (p < 0.05); PaO2 increased from 55 +/- 12 to 257 +/- 148 mmHg 3 h and 270 +/- 136 mmHg 6 h after ALI. (p < 0.05), whereas in controls QS/QT and PaO2 did not improve over the 6 h after onset of ALI. In the LU group mean pulmonary artery pressure was stable for 6 h after ALI (26-29 mmHg), while in controls it increased from 28 +/- 2 to 41 +/- 2 mmHg (p < 0.05). Inhaled LU-135252 reduced cardiac output by 31 +/- 11% (p < 0.05) and increased systemic vascular resistance by 60 +/- 29 % (p < 0.05), while these parameters remained stable in controls. CONCLUSION: In this porcine model of ALI the inhalation of an ET(A) receptor antagonist improved arterial oxygenation and maintained a stable pulmonary artery pressure without inducing systemic vasodilatation.

Overexpression, purification, and characterization of the ADP-ribosyltransferase (gpAlt) of bacteriophage T4: ADP-ribosylation of E. coli RNA polymerase modulates T4 "early" transcription.

The bacteriophage T4 Alt gene product is a component of the phage head and enters the host cell in the process of infection together with the phage DNA. It immediately ADP-ribosylates host RNA polymerase, presumably at only one of the two alpha-subunits. Transcription from T4 "early" promoters, therefore, might be catalyzed, at least in part, by an altered RNA polymerase. The T4 alt gene was cloned into the expression vector pBluescript. E. coli cells, transformed with this recombinant vector, overexpressed the 76 kDa Alt gene product, which was purified to homogeneity. The purified enzyme not only ADP-ribosylates the alpha-subunit of RNA polymerase, but also subunits beta and beta', as well as the sigma 70-factor. The recombinant enzyme behaved like the native enzyme isolated from mature phage particles. The effect of the ribosylation reaction on the transcription activity of host RNA polymerase was investigated in vivo. It results in a modulation of T4 "early" promoter strengths, presumably, in a number of cases, leading to an overexpression of T4 "early" genes. The degree of overexpression, in some cases, should reach 50%, and seems to be well dosed for each promoter, controlling an individual transcription unit.

Changes in magnetic resonance transverse relaxation times of two muscles following standardized exercise.

Magnetic resonance (MR) imaging studies of exercising leg muscles were performed to compare the changes in MR transverse relaxation times (T2) that result from exercise of the anterior tibialis (AT) and extensor digitorum/hallicus longus (E) in the anterior compartment of the lower leg with those T2 changes in the medial and lateral gastrocnemius (G) in the posterior compartment. Spin-echo MR images were obtained at 1.5 Tesla before and during the first 14 min of recovery from dynamic exercise. In order to normalize the exercise, workloads for each subject were set at 25% of the measured maximum voluntary contraction (MVC) of the anterior and posterior compartments. In separate exercise sessions, a nonmagnetic, pneumatic exercise apparatus was employed for either dorsiflexion or plantarflexion against a fixed constant resistance for two different exercise durations (1 min 45 s or 5 min). Transaxial MR images (TR = 1000 ms, TE = 30, 60, 90, 120 ms, 128 x 256 matrix, 1.5 cm slice) were used to calculate T2 values. Although subjects performed approximately 7-fold more work (P < or = 0.001, dorsiflexion vs plantarflexion) during plantarflexion than during dorsiflexion at both exercise duration's, the exercise induced T2, while being greater than those at rest (P < or = 0.001), were not significantly different in the different compartments. We conclude that, when exercised at the same workload (25% of MVC), these two muscles produce T2 changes that are not significantly different from each other.

Heterogeneous signal intensity in magnetic resonance images of hypertrophied left ventricular myocardium.

Left ventricular hypertrophy is associated with decreased longevity and often leads to congestive heart failure. An exploratory study of magnetic resonance imaging in human left ventricular hypertrophy was performed. First, 13 patients with left ventricular hypertrophy and 7 controls of similar ages were studied using electrocardiogramgated end-diastolic images. Visual inspection suggested that low-intensity zones were frequently found within the hypertrophied myocardium. To verify this observation, the images were processed with semi-automatic edge detection and a derivative-based tissue characterization algorithm, yielding tissue heterogeneity indices (THI-A and THI-V) which objectively measured the low-intensity zones. THI-A and THI-V were both significantly greater in left ventricular hypertrophy patients than in controls (THI-A: 0.111 vs 0.038, p = 0.009). THI was also significantly correlated with duration of disease and electrocardiographic abnormalities. To validate these initial findings prospectively, the same quantitative analysis was applied to magnetic resonance images of an additional 20 left ventricular hypertrophy patients and 12 controls from two institutions, using different imaging systems and different acquisition parameters. Again, THI was significantly greater in patients than in controls. Analysis of end-systolic images yielded similar results. In four dogs with left ventricular hypertrophy induced by aortic banding, THI showed a statistically significant increase as left ventricular hypertrophy developed. Hypertrophied myocardium thus shows reproducible differences from normal tissue with magnetic resonance imaging; hence, quantitative magnetic resonance tissue characterization may be useful in assessing pathologic changes in LVH.

ADP-ribosylation and early transcription regulation by bacteriophage T4.

Bacteriophage T4 codes at least for two ADP-ribosylating activities, the 76 kDa Alt and the 24 kDa Mod gene products. The main target for both enzymes is the host RNA polymerase. We cloned and sequenced the alt gene and overexpressed the corresponding enzyme. The recombinant protein shows ADP-ribosylating activities in vitro, as had been described earlier for the native enzyme isolated from phage heads. The native as well as the recombinant protein ADP-ribosylate the alpha-subunit of RNA polymerase, but also subunits beta, beta' and sigma 70 and perform an autoribosylation reaction. Taking advantage of the pKWIII test system, constructed to measure promoter strengths in vivo, it was found that ADP-ribosylation of RNA polymerase leads to an increase of transcription from T4 early promoters up to a factor of two. In an infected host cell this should cause an enhanced expression of T4 genes. Depending on whether RNA polymerase was ADP-ribosylated or not, it initiated transcription at T4 promoters with different sequence characteristics: unribosylated RNA polymerase recognizes the early T4 promoters by an extended -10 region, whereas the ribosylated enzyme selects for T4 early promoters with an extended T4-specific and highly conserved -35 region. These results may reflect how the virus, step by step imposes its genetic program on the host cell, and in part they give a rationale for the extension of the consensus sequence observed with these promoters. We also sequenced the genomic region of the T4 mod gene and found two open reading frames coding both for proteins of approximately 24 kDa. Up to now none of the reading frames could be cloned into E. coli in an active form, making it highly probable that the ADP-ribosylation pattern inflicted by gene product Mod on host RNA polymerase is deleterious to these bacteria. Comparisons of the amino acid sequences showed significant homologies among the two reading frames. Computer analysis reveals that both Mod sequences and also the sequence of the Alt protein exhibit a structural concordance with the catalytic domains of other prokaryotic ADP-mono-ribosyltransferases such as the Pseudomonas aeruginosa exotoxin A, the cholera labile enterotoxin, the diphteria toxin, the heat labile enterotoxin A of E. coli, and pertussis toxin. We present a detailed model for T4 transcription regulation.

Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector.

We report on the construction of promoter probe vector pKWIII, useful in cloning and analyzing strong promoters for Escherichia coli RNA polymerase. Also T4 early promoters that proved to be difficult to clone with other vectors could be tested. The promoter activities obtained with this convenient and nonradioactive system largely correspond to those determined by pulse-labeling of transcripts in the same system. Results with well-characterized control promoters are in good agreement with values given by other authors. We present relative activities of several early promoters of phage T4 and compare these to promoter activities of other phages. Sorting the T4 promoters according to strength suggests the importance of distinct sequence elements to promoter functioning. They are centered around positions -52, -42, and -15.

Computer integration and time spent on direct patient nutritional care activities in an outpatient dialysis setting.

OBJECTIVE: To compare time spent in patient and administrative activities by nutrition professionals in a group of dialysis facilities, and relate differences to the degree of computer integration at the sites and between 2 time periods. DESIGN: Survey design, self-administered activity form, completed in 1992 and 1997. SETTING: Three of the largest outpatient dialysis centers in western Washington state. SUBJECTS: Ten nutrition professionals completed the activity forms. INTERVENTION: Activity form (recording time), questionnaire (results not reported here). MAIN OUTCOME MEASURES: Amount of time spent on administrative activities in 1997 and the number of patients and number of hours per nutrition professional per month spent with patients in 1997, compared with 1992. RESULTS: Time spent on administrative activities decreased in all facilities since 1992, with the largest decrease at the unit having the most computer integration. CONCLUSION: Extensive computer integration seems to allow for a decrease in the amount of time spent on administrative activities by nutrition professionals.

Adverse dermatologic reaction to transdermal nitroglycerin.

The incidence of adverse effects from transdermal nitroglycerin systems is reported to be very low. A cutaneous reaction from Transderm-Nitro is described. The patient in this case was a 63-year-old male with a history of chronic obstructive lung disease, adult onset diabetes mellitus, peptic ulcer disease, congestive heart failure, and angina pectoris. Seven weeks after having used a Transderm-Nitro-5 patch daily, the patient was found to have two macular erythematous areas on his chest that corresponded to the exact sites of the nitroglycerin patch. Follow-up with the same patient, using a placebo Transderm-Nitro, elicited a reaction similar in appearance. The exact causative agent is unknown, but it is believed to be from the transdermal delivery system and not the nitroglycerin.

Motion artifact in T2-weighted fast spin-echo images of the liver: effect on image contrast and reduction of artifact using respiratory triggering in normal volunteers.

The purpose of our study was to evaluate the effect of respiratory motion on the image contrast of T2-weighted fast spin-echo (FSE) images of the liver as well as the reduction of motion artifact using respiratory triggering of the data acquisition. We imaged the livers of 10 healthy volunteers using a fast spin-echo T2-weighted sequence. Images were obtained both without and with patient triggering. Triggered images were acquired in a segmented fashion during multiple sequential breath-holds using an echo train of 8 or 16, both with and without flow compensation (gradient moment nulling). Ratios of signal difference to noise (SD/N) of the liver and gallbladder as well as the liver and spleen were compared for all sequences. All of the triggered images showed statistically significant improvement of SD/N for the liver and gallbladder as well as for the liver and spleen when compared with the nontriggered images. Triggered images obtained with an echo train length of 8 and, with flow compensation, showed the highest SD/N ratios. In one volunteer whose liver contained multiple small cysts, the triggered images showed improved visualization of individual cysts and identified a larger number of cysts. Respiratory motion causes a significant loss of contrast on T2-weighted fast spin-echo images of the liver. This can be reduced by using a segmented data acquisition triggered by the respiratory cycle obtained during sequential breath-holds.

Prevalence of low albumin, suboptimal energy, and muscle stores in asian dialysis patients.

OBJECTIVE: Investigate the prevalence of low serum albumin levels (<3.2 g/L) and decreased arm muscle area percentiles and arm fat area percentiles in Asian patients compared with non-Asian patients treated with dialysis. DESIGN: Cross-sectional study in which serum albumin and anthropometric measurements were averaged over 6 months, and compared between patients of Asian ethnicity and patients of non-Asian ethnicity. SETTING: Eight outpatient dialysis facilities. SUBJECTS: Ninety-seven Asian and 513 non-Asian patients treated with hemodialysis or peritoneal dialysis. RESULTS: Height, weight, and body mass index were significantly lower in Asians compared with non-Asians (P <.001). Protein catabolic rate was significantly greater in Asian (1.17 +/- 0.29 g/kg) compared with non-Asian (0.97 +/- 0.28 g/kg) women (P <.001). Asian men (3.30 g/dL) and women (3. 26 g/dL) had lower serum albumin compared with non-Asian men (3.35 g/dL; P =.057) and women (3.34 g/dL; P =.040). The proportion of patients with serum albumin <3.2 g/dL was greater for both Asian women (35%) (P <.040) and men (30%) than non-Asian women (25%) and men (20%). After adjusting for important covariates, serum albumin remained significantly different between Asian and non-Asian patients (P <.05). The proportion below the fifth percentile for arm muscle area was significantly greater for both Asian men (54%) and women (19%) compared with non-Asian men (24%) and women (8%). Proportions of Asian and non-Asian women below the 10th and 5th percentile for arm fat area were similar. However, Asian men (54%) had a significantly greater fat depletion than non-Asian men (26%). CONCLUSION: Mean serum albumin was significantly lower in Asian patients on dialysis than in non-Asians. Muscle stores were depleted in Asian men and women compared with non-Asians, and fat stores were depleted in Asian men. Based on this study, Asian dialysis patients would seem to be at higher nutritional risk than non-Asians, particularly Asian men. Further research is needed to assess factors that affect serum albumin and mortality in Asian dialysis patients, and standards need to be developed to further assess anthropometric measurements in this population.

Serum albumin concentration-related Health Care Financing Administration quality assurance criterion is method-dependent: revision is necessary.

The objective of this study was to examine quantitative differences between the two commonly used methods for determining serum albumin concentration, bromcresol green (BCG) and bromcresol purple (BCP), in normal subjects and in 235 unselected dialysis patients in view of recently established Health Care Financing Administration (HCFA) quality assurance review criteria. The mean of normal results by the BCG method was 4.4 g/dL, and 97.5% of values were 3.8 g/dL or higher. The mean of normal results by the BCP method was 3.9 g/dL, and 97.5% of values were 3.3 g/dL or higher. Serum albumin concentrations in samples from the dialysis patients had respectively lower mean values by both methods. For the BCG method, the mean was 3.8 g/dL, and 82% of values were 3.5 g/dL or higher; for the BCP method, the mean was 3.3 g/dL, and 82% of values were 3.0 g/dL or higher. Likewise, for the reference immunonephelometric procedure, the mean value for the dialysis patients was 3.3 g/dL, and 82% of values were 3.0 g/dL or higher. For the samples from the dialysis patients, in comparison with the immunonephelometric method, the BCG method exhibited both constant (intercept = 9.3 g/L) and proportional error (slope = 0.87). The mean albumin value for the BCG method was 3.8 g/dL, 15% higher. In contrast, the BCP method compared closely with the reference method: slope = 1.00, intercept = 0.8 g/L, mean x = 3.3 g/dL, mean y = 3.3 g/dL. The HCFA quality assurance criteria are valid only for the BCG method.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of cDNAs encoding human and gibbon GM-CSF.

We have used a mammalian cell expression cloning system to identify cDNA clones encoding human granulocyte-macrophage colony stimulating factor. The human clone was used as a hybridization probe to identify the corresponding sequence from a cDNA library prepared from a gibbon T-cell line. The human cDNA has been used to produce recombinant GM-CSF in monkey COS-1 cells. The purified protein from COS cells is very similar to the GM-CSF isolated from a continuous human T-cell line.