Functional differences among the six Saccharomyces cerevisiae tRNATrp genes

The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes. Other, non-functional tDNATrp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNATrp, as well as a 34-nucleotide intervening sequence, are identical. However, the 5' and 3' flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes. Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 mu vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5'-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3'-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5'-flanking sequences of the tRNATrp genes.     

Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins

Rat hepatocytes de-differentiate and proliferate when cultured on collagen-coated dishes in a chemically defined Hepatocyte Growth Medium in the presence of hepatocyte growth factor and epidermal growth factor. The addition of biomatrix derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma stops this process and leads to re-differentiation of the cells. We monitored DNA binding activity and protein levels of CCAAT/Enhancer Binding Proteins (C/EBPs) during these events by electrophoretic mobility shift assays and western blot analysis. We used plasma protein gene expression as a marker for the proliferation and differentiation phases. During the initial proliferation phase the DNA binding activity of C/EBPs decreased about 5-10 fold, mainly due to reduction of C/EBP alpha protein to nearly undetectable levels. Addition of EHS-gel prevented the further loss of C/EBP alpha protein and established a new steady state level. Since C/EBP beta proteins were affected to a much lesser extent, the C/EBP alpha:C/EBP beta ratio was greater in the presence of EHS-gel. Transferrin, alpha 1-antitrypsin, and albumin mRNA expression increased substantially. Thus stabilized C/EBP alpha expression, an increased C/EBP alpha:C/EBP beta ratio, and increased expression of liver specific mRNAs all correlated with the transition of proliferative to differentiated cells.     

Pyridoxalated hemoglobin polyoxyethylene conjugate does not restore hypoxic pulmonary vasoconstriction in ovine sepsis

OBJECTIVES: Hypoxic pulmonary vasoconstriction, a protective mechanism, minimizes perfusion of underventilated lung areas to reduce ventilation-perfusion mismatching. We studied the effects of sepsis on hypoxic pulmonary vasoconstriction and attempted to determine whether hypoxic pulmonary vasoconstriction is influenced by pyridoxalated hemoglobin polyoxyethylene conjugate, a nitric oxide scavenger. DESIGN: Prospective, randomized, controlled experimental study with repeated measures. SETTING: Investigational intensive care unit at a university medical center. SUBJECTS: Nineteen female merino sheep, divided into three groups: group 1, controls (n = 5); group 2, sheep with sepsis (n = 6); and group 3, septic sheep treated with pyridoxalated hemoglobin polyoxyethylene conjugate (n = 8). INTERVENTIONS: All sheep were instrumented for chronic study. An ultrasonic flow probe was placed around the left pulmonary artery. After a 5-day recovery, a tracheostomy was performed and a double-lumen endotracheal tube was placed. Animals in groups 2 and 3 received a 48-hr infusion of live Pseudomonas aeruginosa (6 x 10(4) colony-forming units/kg/hr). After 24 hrs, sheep in group 3 received pyridoxalated hemoglobin polyoxyethylene conjugate (20 mg/kg/hr) for 16 hrs; sheep in groups 1 and 2 received only the vehicle. Hypoxic pulmonary vasoconstriction was repeatedly tested by unilateral hypoxia of the left lung with 100% nitrogen. Hypoxic pulmonary vasoconstriction was assessed as the change in left pulmonary blood flow. MEASUREMENTS AND MAIN RESULTS: In the animals in group 1, left pulmonary blood flow decreased by 62 +/- 8 (SEM)% during left lung hypoxia and remained stable during repeated hypoxic challenges throughout the study period. After 24 hrs of sepsis, left pulmonary blood flow decreased from 56 +/- 10% to 26 +/- 2% (group 2) and from 50 +/- 8% to 23 +/- 6% (group 3). In the sheep in group 2, there was no adaptation over time. Pulmonary shunt fraction increased. Pyridoxalated hemoglobin polyoxyethylene conjugate had no effect on hypoxic pulmonary vasoconstriction or pulmonary shunt. The animals receiving the bacterial infusion developed a hyperdynamic circulatory state with hypotension, decreased systemic vascular resistance, and increased cardiac output. Pyridoxalated hemoglobin polyoxyethylene conjugate increased mean arterial pressure and systemic vascular resistance but did not influence cardiac index. Pulmonary arterial pressure was increased during sepsis and increased even further after pyridoxalated hemoglobin polyoxyethylene conjugate administration. Oxygenation and oxygen delivery and uptake were not affected by pyridoxalated hemoglobin polyoxyethylene conjugate. CONCLUSIONS: Hypoxic pulmonary vasoconstriction is blunted during sepsis and there is no adaptation over time. It is not influenced by pyridoxalated hemoglobin polyoxyethylene conjugate. Pyridoxalated hemoglobin polyoxyethylene conjugate reversed hypotension and, with the exception of an increase in pulmonary arterial pressure, had no adverse effects on hemodynamics or oxygenation.     

The heart at necropsy in centenarians

Cardiac findings at necropsy are described in 6 patients aged 100 to 103 years of age. Although 4 had considerably narrowed coronary arteries at necropsy, none had apparent clinical evidence of myocardial ischemia or congestive heart failure during life.     

The clinical relevance of the Waterlow pressure sore risk scale in the ICU

OBJECTIVE: To evaluate whether the Waterlow pressure sore risk (PSR) scale has prognostic significance for intensive care patients. DESIGN: A prospective study. SETTING: The surgical intensive care unit (ICU) of the University Hospital Rotterdam. PATIENTS: Data were evaluated from 594 patients who had been admitted to the ICU during the year 1994. METHODS AND RESULTS: Each patient was assessed daily with respect to their Waterlow PSR score and the development of pressure sores in the sacral region. Actuarial statistical methods were used to analyse the predictive value of the risk score. When a patient had a Waterlow PSR score > 25 on admission, the risk of developing a pressure sore was significantly increased compared to patients with a PSR score < 25. After admission, the daily Waterlow PSR scores obtained were significantly associated with the risk of developing a pressure sore. For each additional point this risk increased by 23% (95% confidence interval 17 to 28%). CONCLUSIONS: The Waterlow PSR scale provides the medical and nursing staff at an early stage with reliable information about the risk patients have in developing a pressure sore.     

Plasma concentrations of risperidone and its 9-hydroxy metabolite and their relationship to dose in schizophrenic patients: simultaneous determination by a high performance liquid chromatography with electrochemical detection

A simple, sensitive and accurate method for the simultaneous determination of risperidone (RSP) and its 9-hydroxy metabolite (9-OH-RSP) in human plasma is described. The relationship between dose of RSP and the plasma concentration of RSP and 9-OH-RSP in a clinical situation is discussed. Both compounds were isolated from plasma by a simple one-step liquid-liquid extraction with 15% methylene chloride in pentane. High-performance liquid chromatography separations were made on a cyano column and the compounds were detected by electrochemical detector. The method had sufficient sensitivity to determine RSP and 9-OH-RSP accurately at concentrations as low as 0.25 ng/ml when 1 ml of plasma is used for the analysis. The assay determinations were accurate, precise and consistent with a coefficient of variation less than 15%. Commonly co-administered drugs and other antipsychotics did not interfere with the analysis of either RSP or 9-OH-RSP There were large variations in inter- and intra-individual values of plasma concentrations of RSP and 9-OH-RSP. The 9-OH-RSP appears to be the major circulating active moiety and its plasma concentrations were, on the average 22 fold higher than that of RSP in schizophrenic patients treated with RSP. The ratio of RSP/9-OH-RSP concentrations suggested that three of the patients may have deficiency in cytochrome P450 enzyme CYP 2D6. The plasma concentrations of RSP showed a weak relationship with the administered daily oral dose (r = 0.4684, p = 0.01, n = 215). However, there was a good relationship between the daily dose of RSP and the plasma concentration of 9-OH-RSP (r = 0.6654, p = 0.01, n = 280) or the total active moiety, sum of RSP and 9-OH-RSP concentrations (r = 0.7041, p = 0.0005, n = 280). The measurement of the total active moiety in plasma of schizophrenic patients may be useful for assessing the relationship between dose and plasma concentration and dose and clinical outcome of patients rather than measuring RSP alone.     

A review of new oncotropic tracers for PET imaging

We have developed three biochemical probes to determine if they are sensitive probes of early biochemical change in a tumor. All three probes appear to have the appropriate properties for in vivo imaging, but must now be evaluated as probes for the sensitive detection of changes in early malignant disease.     

Bovine plasma oestrogens, progesterone and glucocorticoids during dexamethasone induced parturition

Plasma samples were collected from jugular, uterine and utero-ovarian veins during glucocorticoid induced parturition. Plasma oestrogens, corticosteroids and progesterone were determined by competitive protein binding methods. Corticosteroids and progesterone began to decline within 8 to 10 h following DXMS treatment. Corticoids were only temporarily suppressed, while progesterone fell to minimum levels and remained low through calving. At this stage of gestation (270 days) peripheral plasma progesterone was primarily of ovarian origin. Pre-treatment with HCG appeared to support progesterone production by the CL despite DXMS treatment in 2 of 6 cows. These 2 cows failed to calve within the expected 96 h after DXMS. Plasma oestrogens did not show significant increased until 24 h after DXMS treatment. Cows which responded to DXMS treatment (calved) had significantly higher oestrogen levels than those which did not respond. It was concluded that oestrogens probably play a permissive rather than an initiating role in parturition.