Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressure.

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75-100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with approximately 20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.

Are all diseases infectious?

The complex interactions between microorganisms and human hosts include the well-known, traditional infectious diseases and the symbiotic relation we have with our normal flora. The media have brought to the public's attention many newly described infectious diseases, such as Ebola virus hemorrhagic fever, that were not part of common medical parlance a decade ago. While flooding us with interesting and often dramatic reports of so-called emerging infectious diseases, the media have largely ignored a more fundamental change in our appreciation of human-microorganism interactions: the discovery that transmissible agents may play important roles in diseases not suspected of being infectious in origin. A well-known example is ulcer disease; other examples include neurodegenerative disease, inflammatory disease, and cancer. These fascinating instances of host-pathogen interaction open new prospects for the prevention of disease through immunization.

An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

Loss of neonatal hypoxia tolerance after prenatal nicotine exposure: implications for sudden infant death syndrome.

Maternal cigarette smoking has a high correlation with sudden Infant Death Syndrome, a condition in which cardiorespiratory failure occurs during an hypoxic episode, as in sleep apnea. Pregnant rats were given nicotine infusions of 2 or 6 mg/kg/day throughout gestation, regimens that produce plasma nicotine levels spanning the range in smokers. The day after birth, animals in the high dose group displayed excessive mortality during hypoxic challenge. These animals were found to be deficient in an essential response component, namely adrenomedullary catecholamine release that is required to maintain neonatal cardiac rhythm during hypoxia; the defect was in adrenal cell function rather than in altered innervation or nicotinic receptor desensitization. We also examined brainstem and forebrain noradrenergic mechanisms that are involved in neonatal respiratory control. The nicotine group showed suppressed spontaneous neuronal activity, but were hyperresponsive to hypoxia. As these projections are inhibitory for respiration, the nicotine-induced sensitization would be expected to contribute to respiratory arrest during hypoxia. Prenatal nicotine exposure may thus provide a useful animal model with which to study the physiological mechanisms that underlie Sudden Infant Death Syndrome, while at the same time providing a biological explanation for the association of the syndrome with smoking.

Characterization and molecular analysis of nondisjunction in 18 cases of trisomy 21 and leukemia.

We recently began a cytogenetic and molecular study of nondisjunction in leukemic Down syndrome individuals to determine whether the mechanism by which the extra chromosome 21 originates predisposes the individual to leukemia. In the present report, we summarize our observations on 18 patients with trisomy 21 and acute or transient leukemia, including 11 patients with acute lymphocytic leukemia, three with acute myeloid leukemia, one with B-cell lymphoma, one with acute megakaryoblastic leukemia, and two with transient leukemia. Results of DNA marker studies of the parental origin of the extra chromosome 21 indicated that 16 of the 18 cases (89%) were maternally derived, a percentage similar to that seen among nonleukemic Down syndrome patients. We noted that most leukemic Down syndrome patients had one locus or more in which parental heterozygosity was maintained in the trisomic individual, indicating a meiotic rather than a mitotic origin for the trisomy.

Infections complicating mycosis fungoides and Sézary syndrome.

OBJECTIVE: To determine, in patients with mycosis fungoides and Sézary syndrome, the incidence of infections, the importance of nosocomial infections, and the epidemiologic factors associated with cutaneous and visceral infections. DESIGN AND SETTING: Retrospective inception cohort study at a university medical center referral clinic. PATIENTS: Three hundred fifty-six patients with mycosis fungoides or Sézary syndrome. MAIN OUTCOME MEASURES: Incidence rates for specific infections, and multivariate risk ratios for demographic and clinical factors associated with infection. RESULTS: Cutaneous bacterial infection was most common (17.0 infections per 100 patient-years), followed by cutaneous herpes simplex virus and herpes zoster virus infection (3.8 infections per 100 patient-years), bacteremia (2.1 infections per 100 patient-years), bacterial pneumonia (1.7 infections per 100 patient-years), and urinary tract infection (1.4 infections per 100 patient-years). Twenty-seven percent of herpesvirus infections disseminated on the skin but none disseminated to internal organs. Pneumonia or bacteremia was present in 88% of patients who died of infection. Only three patients had invasive fungal or protozoal infection. Nosocomial infections accounted for 19% of cutaneous bacterial infections, 59% of bacteremias, 62% of pneumonias, and 88% of infections leading to death. By logistic and Cox regression, the presence of extracutaneous involvement with lymphoma was the most important independent risk factor for recurrent bacterial skin infection (risk ratio [RR], 12; 95% confidence interval [CI], 1.2 to 120), disseminated herpesvirus infection (RR, 28; 95% CI, 2.7 to 290), bloodstream infection (RR, 5.5; 95% CI, 1.7 to 18), and death from infection (RR, 15; 95% CI, 3.6 to 64). CONCLUSIONS: Community-acquired bacterial skin infections are a common cause of morbidity in patients with mycosis fungoides and Sézary syndrome but are usually treated without hospital admission. Bacteremia and pneumonia, which are usually nosocomial, are the major infectious causes of death. Advanced disease stage, independent of corticosteroids and other therapies, is the most important risk factor for both cutaneous and systemic infections.

Expression of mammalian tyrosine aminotransferase in Saccharomyces cerevisiae and Escherichia coli. Purification to homogeneity and characterization of the enzyme overproduced in the bacteria.

Rat liver tyrosine aminotransferase has been expressed in Saccharomyces cerevisiae and Escherichia coli. In yeast, the extent of production is 20-fold higher than that in rat liver after induction by dexamethasone, and reaches 250-fold higher in an E. coli strain carrying the T7 RNA polymerase transcription system. About 250 mg pure and homogeneous enzyme was obtained from 50 g transformed E. coli cells. Determination of Mr and pI, as well as analysis of N- and C-terminal amino acids, suggest that the isolated protein is native. The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post-translational modifications in the mammalian cells are not essential for activity. Pyridoxal 5'-phosphate strongly protects the enzyme against thermal inactivation. After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5'-dithiobis(2-nitrobenzoic acid) whereas only five or six are accessible under native conditions. Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5'-phosphate partially protects them in the holoenzyme. The results are interpreted in the light of the structure/function relationship in this enzyme.

Hot tubs, sex, sushi, and infectious diseases.

Over the past 20 years, striking and interesting changes in patterns of diseases have occurred due to transmissible agents. Among them are the recognition of new diseases, new clinical manifestations of old diseases, new ecologic niches for traditional pathogens, and new modes of disease transmission. Implicated causes for these changing patterns include alterations in lifestyle with respect to sexual behavior, leisure activity, and dietary trends, together with the impact of immigration and the effects of medical progress. A review of these changes demonstrates the dynamic nature of medicine, and the impact that societal change can have on disease patterns.

Early diagnosis of Nocardia asteroides endophthalmitis by retinal biopsy: case report and review.

Hematogenous nocardial endophthalmitis is a rare but devastating infection. Of a total of 10 cases (one described for the first time and nine reported previously in the English-language literature), the lung appeared to be the primary focus of infection in eight (80%). Six (60%) of the cases occurred in individuals receiving corticosteroid therapy; these individuals had undergone renal transplantation (two cases) or cardiac transplantation (one case) and had underlying conditions that included lymphoma (two cases) and chronic active hepatitis (one case). In two immunocompetent individuals, infection followed dissemination from traumatic wounds. Common clinical findings were a rapid decrease in visual acuity and eye pain. All nine of the previously reported cases resulted in total blindness of the involved eye; five patients died not long after diagnosis. In the present report (the first in a cardiac transplant recipient), a favorable outcome with restoration of vision followed early diagnosis through the recently developed technique of fine-needle retinal biopsy.

Antipyretic orders in a university hospital.

PURPOSE: Antipyretics are prescribed for many hospitalized patients, but details concerning prescribing practices are not known. This study was designed to determine the incidence and format of antipyretic orders in a university-based tertiary-care center, and to ascertain whether orders are correlated with patient characteristics or hospital services. PATIENTS AND METHODS: The records of 300 randomly selected patients on the medicine, general surgery, neurosurgery, and obstetrics and gynecology services, and of 75 patients admitted with pneumonia and fever were retrospectively reviewed using a standardized data form. RESULTS: Orders for acetaminophen prn (as needed), without further explanation, were interpreted by the nursing staff as antipyretic orders; 78% of patients with such an order and fever received acetaminophen during the febrile episode. If orders of this type were included, 153 (51%) of the randomly selected patients received an antipyretic order. Gender, age, duration of hospitalization, intensive care unit residence, fever, and presence of a condition worsened by fever were not significant independent predictors of antipyretic prescription, but documented infection and hospitalization on the medicine and neurosurgery services were, with adjusted odds ratios of 2.5 (95% confidence interval [CI] 1.3 to 5.0), 9.4 (95% CI 3.6 to 25), and 14 (95% CI 5.0 to 41), respectively. Of patients who received an antipyretic order, 70% had an admission order for antipyretics; 26%, an order prompted by fever; and 79%, an order while afebrile. In 86%, the order was written prn without further explanation. Around-the-clock dosing, automatic stop orders, and acknowledgement and justification of orders were rare. CONCLUSION: Antipyretic orders are routine and correlate more strongly with hospital service than with individual patient characteristics. They are umprecisely written and generally leave decisions about antipyretic administration to the complete discretion of the nursing staff.

Comparative efficacy and safety of ceftizoxime, cefotaxime and latamoxef in the treatment of bacterial pneumonia in high risk patients.

One hundred and thirty-five patients with bacterial pneumonia who had risk factors (alcoholism, chronic obstructive pulmonary disease, corticosteroid therapy diabetes mellitus, advanced age, solid tumours) were randomly allocated in a double-blind fashion to receive either ceftizoxime (2-4 g every 8 h), cefotaxime (1-2 g every 4 h), or latamoxef (2-4 g every 8 h). Of the 84 patients evaluable for efficacy, clinical cure was achieved in 91%, 85%, and 89% of ceftizoxime- (20/22), cefotaxime-(23/27), and latamoxef-treated (31/35) patients, respectively. Adverse reactions occurred in one of 45 ceftizoxime-treated patients, one of 43 cefotaxime-treated patients, and seven of 47 latamoxef-treated patients. Abnormal laboratory values during therapy were seen in 50% of latamoxef-treated and 43% of cefotaxime-treated patients and in 29% of ceftizoxime-treated patients. Hypoprothrombinaemia occurred in five latamoxef-treated patients and one of these patients experienced an episode of haematemesis. In this study, ceftizoxime, cefotaxime, and latamoxef were similarly effective; however, the incidence of side effects was most frequent with latamoxef.

Brain abscess due to Listeria monocytogenes: case report and literature review.

Listeria monocytogenes is an uncommon cause of brain abscess. Of a total of 14 cases of L. monocytogenes brain abscess (one described for the first time and 13 reported previously in the English-language literature), seven (50%) occurred in patients with leukemia and recipients of renal transplants; four (29%) of the cases occurred in previously healthy individuals. Common clinical findings were similar to those in brain abscess due to other causes and included fever (57%), headache (57%), and focal neurologic signs (64%). Distinctive, however, was the unusually high frequency of associated meningitis and bacteremia; blood cultures were positive in all eight cases in which they were performed. Eight (57%) of the 14 patients died. L. monocytogenes should be included in the differential diagnosis of brain abscess in patients with leukemia and in renal transplant recipients. Listerial brain abscess is highly unlikely when blood culture results are negative.

Branhamella catarrhalis pneumonia and immunoglobulin abnormalities: a new association.

A case of Branhamella catarrhalis pneumonia in a patient with multiple myeloma is reported. With the inclusion of this case, 5 of 17 (29.4%) reported cases of Branhamella pneumonia or empyema have occurred in patients with underlying diseases associated with immunoglobulin abnormalities. This strong clinical association suggests that qualitatively and quantitatively normal immunoglobulins are important host defense mechanisms in preventing infection with this pathogen. The sputum Gram stain demonstrating kidney-shaped gram-negative diplococci may be an early clue to the diagnosis, as well as an initial guide to empiric therapy, and may help the laboratory isolate and identify this pathogen, which, because of its morphologic resemblance to Neisseria, is frequently reported as "normal flora".

Anterior sacral meningocele: report of five cases and review of the literature.

Five new cases of anterior sacral meningocele are presented, including one secondary to neurofibromatosis, a previously undescribed association. The literature is reviewed, drawing attention to the relationship between anterior sacral meningocele, sacral dysgenesis, and other congenital anomalies. Special consideration is given to the clinical features of this entity, as well as to the techniques and results of surgical management.