Cooperation between the Cdk inhibitors p27KIP1 and p57KIP2 in the control of tissue growth and development

Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and differentiation in several tissues. Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdk activity to activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, we show that the Cdk inhibitors p27^KIP1 and p57^KIP2 function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27^KIP1 and p57^KIP2 are critical terminal effectors of signal transduction pathways that control cell differentiation.     

Comparison of two automated DNA amplification systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis.

Eighty-four specimens of respiratory secretions culture positive for mycobacteria (70 positive for Mycobacterium tuberculosis and 14 positive for nontuberculous mycobacteria) and 120 culture-negative specimens were evaluated by three DNA amplification techniques: a manual in-house single-tube nested PCR (nPCR) and two commercial automated assays (the Cobas Amplicor System [aPCR-h] from Roche Diagnostic Systems and the Abbott LCx Probe System [aLCx-p] from Abbott Laboratories). The overall diagnostic sensitivities of the nPCR, aPCR-h, and aLCx-p were 77.1, 84.3, and 77.1%, respectively, and the sensitivities were 57.9, 57.9, and 36.8%, respectively, for smear-negative specimens. Specimens culture positive for nontuberculous mycobacteria were negative by all three assays. Eight culture-negative specimens which were positive by one or more assays had previously been documented by culture to be positive for M. tuberculosis and were taken from patients who were treated with antituberculosis agents. Retesting of specimens negative by one assay by the other two assays revealed that each test had its unique group of negative specimens. When considering the DNA extraction and amplification steps of these assays separately, it was found that extracts from aPCR-h and aLCx-p were compatible with nPCR amplication, while the two automated assays could only amplify extracts processed with their own reagents. Limiting dilution analysis revealed that the order of analytical sensitivity was nPCR, followed by aLCx-p and then aPCR-h. Comparison of the work flow of each assay revealed that although the aPCR-h demands the least specimen handling, the turnaround time of aLCx-p is the most favorable.     

Reactive hemophagocytic syndrome: a study of 7 fatal cases

Reactive hemophagocytic syndrome is a clinico-pathologic entity characterized by systemic proliferation of non-neoplastic histiocytes showing phagocytosis of hemopoietic cells, resulting in blood cytopenia. It is best known to be associated with virus infection, but other associated diseases have also been implicated. The clinical and pathological findings of 7 fatal cases are described. The syndrome affected both sexes of a wide age range, and all patients had fever. Significant laboratory findings were blood cytopenia, abrupt drop in the blood cell counts, deranged liver function tests and abnormal coagulation profile. The associated diseases were diverse: two patients had bacterial infection; two had peripheral T-cell lymphoma; one had disseminated undifferentiated carcinoma of the ovary; one had both tuberculosis and disseminated nasopharyngeal carcinoma, and one had no obvious underlying disease. It is postulated that lymphokines secreted by lymphoid cells or tumor cells may be responsible for the systemic activation of histiocytes. The differential diagnosis from malignant histiocytosis is discussed.     

Evaluation of four methods for detection of group B streptococcal colonization.

Four methods (streak plate, pour plate, selective broth, and direct fluorescent-antibody staining) were evaluated for their ability to detect group B streptococcal colonization in parturient women and their offspring. When colonization was defined as a positive culture by any method from any site, selective broth was the most sensitive method, detecting 100% of colonized mothers and infants at birth and 48 h of age. This method failed to detect only one colonized individual (infant at 24 h of age). The other three methods detected from 20 to 56% of colonized individuals.     

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.     

Comparison of three different sensitive assays for hepatitis B virus DNA in monitoring of responses to antiviral therapy

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log(10) HBV DNA levels (6.66 versus 7. 17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log(10) HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6. 3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment.     

Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction.

Two major antigens of Bordetella pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (PT), were efficiently purified from culture filtrate by exploiting their relative hydrophobicities and differences in affinity to sialic acid-containing protein. High yields of FHA (40 to 80 mg/liter) and PT (8 to 16 mg/liter) were first produced by growing the bacteria in the modified CL medium. The FHA and PT in the culture filtrate were adsorbed onto butyl-Sepharose by hydrophobic interaction at appropriately high ionic strength. Elution of the antigens was effected by decreasing their hydrophobicities with a buffer of low ionic strength. FHA was then separated from PT with an affinity column of fetuin-Sepharose. The fraction passing through the column contained purified FHA, and the fetuin-bound PT was eluted with buffered MgCl2. The FHA and PT purified by these steps were electrophoretically and serologically identical to the reference purified FHA and PT preparations. Approximately 16 to 32 mg of purified FHA and 4 to 8 mg of purified PT were obtained from 1 liter of culture filtrate. The described procedure for making FHA and PT antigens from B. pertussis for serologic and immunologic use is very simple, efficient, and reproducible.     

Class II defective avian sarcoma viruses: Comparative analysis of genome structure

The genomes of class II avian sarcoma viruses PRCII, PRCII-p, PRCIV, and Fujinami sarcoma virus (FSV), were studied by oligonucleotide fingerprinting, heteroduplex mapping, and nucleic acid hybridization. All of these viruses are genetically defective and have a small RNA genome between 4.5 and 6.1 kilobases (kb) in length. They contain helper-related sequences at both the 5′- and 3′-ends, but most of the retroviral sequences in the middle of the genome are deleted. In place of this deleted information, a contiguous stretch of transformation-specific sequences, termed fps, is found. These putative oncogenic sequences are about 1.2 kb in PRCII, and those in PRCII-p and PRCIV are roughly 2.9 kb. From the analysis of oligonucleotides, it appears that the fps sequences of PRCII represent a subset of those of PRCII-p. Most of the additional sequences present in PRCII-p but absent from PRCII are at the 5′-half of fps. The helper-related sequences in PRCII and PRCII-p are almost indistinguishable, except that PRCII-p contains slightly more retroviral information at the 3′-end of the genome. Therefore, it is possible that PRCII has been derived by deletion from PRCII-p. By contrast, PRCII-p and PRCIV were found to contain identical fps sequences, but their helper-related sequences have diverged substantially. These two sarcoma viruses either represent two independent isolates or, if derived from a single isolate, they have undergone extensive mutation and recombination with diverse avian retroviruses. FSV was found to differ to a greater extent from other class II sarcoma viruses in both helper-related and fps sequences. The difference in fps sequences is localized in the 5′-half of that region. Considering the variation in fps among all members of class II avian sarcoma viruses, it appears that the 3′-half of that genetic region is more conserved than the 5′-half.     

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.