Functional characterization of the early and late mRNAs of simian virus 40.

We have characterized the early SV40 mRNAs by size fractionation on acrylamide gels followed by in vitro translation and immunoprecipitation. The mRNA for the 17K T antigen migrates more slowly than that for the 100K T antigen, with the apparent size difference being 250 nucleotides This finding is consistent with the mRNA for the 17K T antigen corresponding to an almost complete copy of the early region while the mRNA for the 100K T antigen would lack the sequences between 0.59 and 0.54 map unit. By in vitro translation of SV40-specific RNA followed by immunoprecipitation we have been unable to detect the synthesis of any T antigen intermediate in size between the 100K and 17K T antigens. We have demonstrated the synthesis of two proteins from intracellular SV40-specific mRNAs which appear to be identical to the VP2 and VP3 found in the virion on the basis of their mobilities in acrylamide gels and their tryptic peptide maps. Both VP2 and VP3 are labeled with formyl-[³⁵S]methionine when synthesized in the presence of [³⁵S]fMet-tRNA_f^Met. By analyzing partial proteolysis products of such N-terminally labeled proteins we could show that VP2 and VP3 share common C-termini. Therefore, VP3 is initiated and made independently of VP2 and is not derived from VP2 by proteolytic processing. VP2 and VP3 are both made from mRNAs about 19 S in size. The mRNA for VP2 migrates marginally more slowly than the mRNA for VP3 in acrylamide gels.     

Problems affecting performance of the fluorescent treponemal antibody-absorption test for syphilis.

Immunofluorescent staining of Treponema pallidum was studied to clarify the effect of three factors on the results of the fluorescent treponemal antibody-absorption test: (i) heat inactivation of sera at 56 degrees C for 30 min before testing, (ii) use of multicircle slides, and (iii) tungsten illumination to visualize and assess unstained treponemes on reactive as well as nonreactive smears. It was found that serum inactivation before testing was not necessary for detection of immunoglobin G antibody, but an immunoglobulin M prozone was detected in unheated serum. On multicircle slides, it was demonstrated that a false-positive reaction could be obtained in 30 s at 37 and 25 degrees C if a smear where a nonreactive serum had been placed was crossed by a strongly reactive serum from another circle. Tungsten illumination proved necessary for correct assessment of unstained treponemes on all fluorescent treponemal antibody-aborption test smears, reactive or nonreactive. The possible role of these factors in incorrect fluorescent treponemal antibody-absorption test results is discussed.     

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants

Summary The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.     

Interaction between a peroxisome proliferator-activated receptor gamma gene polymorphism and dietary fat intake in relation to body mass

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a critical regulator of adipogenesis. PPAR gamma+/- mice are resistant to high-fat diet-induced obesity and thus PPAR gamma may mediate physiological responses to dietary fat in other mammals. The aim of this study was to determine whether the human PPAR gamma proline to alanine substitution polymorphism (Pro12Ala) modifies the association between dietary fat and adiposity and plasma lipids. Subjects (n=2141) were controls selected for three case-control studies nested within the Nurses' Health Study, a large ongoing prospective cohort study. Associations between intake of total fat, fat subtypes and BMI were different in PPAR gamma 12Ala variant allele-carriers compared with non-carriers. Among homozygous wild-type Pro/Pro individuals, those in the highest quintile of total fat intake, had significantly higher mean body mass index (BMI) compared with those in the lowest quintile (27.3 versus 25.4 kg/m2, respectively; P-trend<0.0001) whereas among 12Ala variant allele-carriers there was no significant trend observed between dietary fat intake and BMI (P-trend=0.99; P-interaction=0.003). In contrast, intake of monounsaturated fat was not associated with BMI among homozygous wild-type women but was inversely associated with BMI among 12Ala variant allele-carriers (mean in lowest quintile=27.6 versus mean in highest quintile=25.5 kg/m2; P-trend=0.006; P-interaction=0.003). The relationship between dietary fat intake and plasma lipid concentrations also differed according to PPAR gamma genotype. These data suggest that PPAR gamma genotype is an important factor in physiological responses to dietary fat in humans.     

Two Methods for Identifying Wiener Cascades Having Noninvertible Static Nonlinearities

Two methods are proposed for identifying the component elements of a Wiener cascade that is comprised of a dynamic linear element (L) followed by a static nonlinearity (N). Both methods avoid potential problems of instability in a procedure presented by Paulin [M. G. Paulin, Biol. Cybern. 69: 67–76, 1993], which itself is a modification of a method described earlier by Hunter and Korenberg [I. W. Hunter and M. J. Korenberg, Biol. Cybern. 55: 135–144, 1996]. The latter method is a rapidly convergent iterative procedure that produces accurate estimates of the L and N elements from short data records, provided that the static nonlinearity N is invertible. Subsequently, Paulin introduced a modification that removed this limitation and enabled identification of Wiener cascades with nonmonotonic static nonlinearities. However, Paulin presented his modification employing an autoregressive moving average (ARMA) model representation for the dynamic linear element. To remove the possibility that the estimated ARMA model could be unstable, we recast the procedure by utilizing instead a rapid method for finding an impulse response representation for the dynamic linear element. However, in this form the procedure did not have good convergence properties, so we introduced two key ideas, both of which provide effective alternatives for identifying Wiener cascades whether or not the static nonlinearities therein are invertible. The new procedures are illustrated on challenging examples involving high-degree polynomial static nonlinearities, of odd or even symmetry, a high-pass linear element, and output noise corruption of 50%. © 1999 Biomedical Engineering Society. PAC99: 8710+e, 0210Nj, 0250-r     

Mantle cell lymphoma

OBJECTIVE: This article summarizes the most useful ancillary immunohistochemical and molecular assays for use in the diagnosis of mantle cell lymphoma. DATA SOURCES: The English language literature was surveyed, with an emphasis on recent publications, for articles presenting key advances in the molecular characterization of mantle cell lymphomas and for series of cases testing the utility of molecular diagnostic tests. The authors' series of 26 small B-cell lymphomas, analyzed for the cyclin D1 protein by paraffin immunohistochemistry and for t(11;14) by polymerase chain reaction, is included. CONCLUSIONS: Mantle cell lymphoma, a B-cell lymphoma now recognized in the 1994 Revised European-American Classification of Lymphoid Neoplasms (REAL) classification, is a relatively aggressive lymphoma with a poor prognosis. Its characteristic t(11;14)(q13;q32) translocation has a role in oncogenesis and has been exploited for molecular diagnostic tests, but these tests vary in sensitivity, specificity, and ease of use. Improved immunohistochemical tests are sufficient to confirm the diagnosis in most cases. Conventional cytogenetics and molecular diagnostic tests for t(11;14)-Southern blot and polymerase chain reaction analysis-may be helpful in selected cases, but are laborious or of limited sensitivity. Other methods, such as fluorescence in situ hybridization, need further development to provide faster, more sensitive diagnosis.     

Detection of immunoglobulin M in cerebrospinal fluid from syphilis patients by enzyme-linked immunosorbent assay.

Cerebrospinal fluid (CSF) samples were evaluated in an immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) for syphilis with sonic extracts of Treponema pallidum coated on polystyrene plates. The ELISA procedure was reproducible, and T. pallidum antigens were stable., A total of 15 CSF samples from patients with neurosyphilis, 18 CSF samples from patients with syphilis, 12 CSF samples from patients treated for syphilis, and 494 CSF samples from patients with neurologic or other systemic diseases were tested. The IgM ELISA gave reactive results in all of six symptomatic and congenital neurosyphilitic patients and none of nine asymptomatic neurosyphilitic patients. Of 524 CSF samples from nonneurosyphilitic individuals, 513 were nonreactive, resulting in 98% test specificity. The IgM ELISA in CSF should prove to be useful for confirmation of symptomatic neurosyphilis.     

Growth and development in thanatophoric dysplasia

Two cases of prolonged survival of thanatophoric dysplasia are presented, in which ventilatory support was initiated in the neonatal period because of respiratory distress. Both patients required a ventriculoperitoneal shunt for hydrocephalus and had decompression of the posterior fossa. The history of each patient has been characterized by profound developmental delay and dramatic growth failure.     

A field-suitable, semisolid aerobic enrichment medium for isolation of Campylobacter jejuni in small numbers

The objective of this study was to produce an economical, easy to prepare, field-suitable enrichment medium for detection of Campylobacter jejuni in small numbers. A semisolid aerobic enrichment medium was developed. Rates of recovery from inoculated medium, sterile swabs, and mixed cultures of C. jejuni and coliform bacteria were tested.