Use of a histone H4 promoter to drive the expression of homologous and heterologous proteins by Penicillium funiculosum.

Two genes encoding histone H4 (H4.1 and H4.2) from Penicillium funiculosum have been cloned and characterised. Structurally, the histone H4.1 gene is divergently linked to the histone H3 gene and the two genes are separated by approximately 800 bp. The transcription of the histone H4.1 and H4.2 genes in P. funiculosum appears to be distinctively regulated. Histone H4.1 mRNA showed a high steady-state level during the early stages of batch culture that decreased as growth reached the stationary phase. In contrast, the expression of the histone H4.2 gene was lower than that of H4.1 throughout batch growth and increased gradually with time. In order to expand the industrial application of P. funiculosum as a host for the production of heterologous proteins, the promoter of the histone H4.1 gene was successfully used to drive the expression of an intracellular bacterial enzyme, beta-glucuronidase, and a secreted homologous enzyme, xylanase C. The constitutive secretion of xylanase C was achieved in the absence of other xylanases by batch fermentation in the presence of glucose.

A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module.

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.

Effect of hormone variations and other factors on symptom severity in women with dystonia.

OBJECTIVE: To determine if any relationship exists between the severity of symptoms in women with dystonia and female reproductive hormonal variations. PATIENTS AND METHODS: We surveyed 279 women with dystonia seen at Mayo Clinic Scottsdale over a 6-year period (1990-1995), and 204 responded. The women were asked questions regarding their reproductive and menstrual histories and dystonia severity and other questions with an emphasis on possible exacerbating or relieving factors. RESULTS: Although in the majority of women hormonal influences had no consistent effect on dystonia symptom severity, 26 (41.9%) of 62 premenopausal women noted a change in the severity of their dystonic symptoms in relation to the 3 phases of their menstrual cycle. Other factors that exacerbated dystonia included stress and fatigue, while sleep improved symptoms. Pregnancy, menopause, and hormone replacement therapy had no effect on symptoms. CONCLUSIONS: Menstrual cycling may result in subjective worsening of dystonia symptoms in some women with dystonia. Further clinical and physiologic evaluation is indicated in such patients, as they may represent an important subgroup of dystonic patients that might yield some clues to the pathophysiology of dystonia and to improved treatment strategies.

Barium enema: detection of colonic lesions in a community population.

OBJECTIVE: The purposes of this study were to assess the prevalence of colonic lesions detected at barium enema in a community practice, to compare the findings at barium enema between patients who are asymptomatic and have no known risk factors for colorectal cancer (screening group) and patients who have symptoms of colonic disease or have known risk factors, and to determine if a questionnaire about symptoms and risk factors is an appropriate screening tool. SUBJECTS AND METHODS: A self-administered questionnaire about colorectal symptoms and risk factors was given to 1779 patients scheduled for barium enema examination. On the basis of their responses, patients were divided into three groups: screening group (asymptomatic, without risk factors), symptomatic, and asymptomatic with risk factors. Each patient underwent a fluoroscopic barium enema. We then compared the results (number, histologic type, size of lesion(s), location in the colon, and Patient's age) and risk factors among the three groups. RESULTS: At least one lesion within the colorectum was found in 166 (9%) of 1779 patients at combined proctosigmoidoscopy and barium enema. The prevalence of lesions in the 111 patients with at least one lesion above the rectum at barium enema was 4% (32 of 738) for the screening group, 8% (38 of 476) for asymptomatic patients with risk factors, and 7% (41 of 565) for symptomatic patients (p = .015 when comparing the prevalence in the screening group with the prevalences in the other two groups). Twenty-nine percent of all colonic lesions were found in the screening group. Among the asymptomatic patients, risk factors that included a history of colorectal polyps and advanced age were associated with a significantly higher prevalence of colonic polyps found at barium enema. In the symptomatic group, if patients with histories of polyps were excluded, we were unable to identify other risk factors that led to a significantly higher prevalence of polyps. CONCLUSION: Asymptomatic patients without known risk factors have a significantly lower prevalence of colonic polyps than either symptomatic patients or patients with risk factors alone. Despite this lower prevalence, 29% of all lesions in our series were in the screening group. Assessment of risk factors through a patient questionnaire was not helpful in identifying a group of patients with a higher prevalence of lesions--except for a history of polyps. Management decisions based on a patient questionnaire should be approached with caution. When low-risk patients are denied screening examinations, a significant number of lesions will be missed.

Central venous oxygen saturation, arterial base deficit, and lactate concentration in trauma patients.

Our object was to explore the usefulness of central venous oxygen saturation, arterial base deficit, and lactate concentration in the evaluation of trauma patients. In busy urban trauma centers, limited operating room availability may necessitate that certain hemodynamically stable patients experience some delay between diagnosis of injury and surgery. Because hemodynamic compromise may occur before operation is undertaken, some means of identifying those patients who have the most severe injuries or who are at greatest risk for hemodynamic instability would be useful. We prospectively studied 40 patients with operative truncal injuries admitted to the Cook County Trauma Unit, Chicago, to examine the usefulness of postresuscitation central venous oxygen saturation (ScvO2), arterial lactate concentration, and arterial base deficit in this regard. Preoperative hypotension occurred in 12.5 per cent of these initially stable patients. ScvO2 did not significantly correlate with any of the parameters of blood loss and severity of injury examined. However, both base deficit and lactate concentration correlated with transfusion requirements; in addition, base deficit correlated with trauma score, and lactate correlated with peritoneal shed blood volume. Our data suggest that, after resuscitation, arterial base deficit and lactate concentration may be better indicators of blood loss than is ScvO2.