Sequence diversity of human rotavirus strains investigated by northern blot hybridization analysis.

Rotavirus genomic RNAs, derived from a series of human isolates that exhibit variability in the pattern of migration of the double-stranded RNA on polyacrylamide gels, were transferred to diazobenzyloxymethyl paper, and their sequence diversity was investigated. Hybridization of cDNA probes prepared from the 11 segments of rotavirus RNA indicated that considerable sequence diversity exists among these viruses. Under conditions of both low and high stringency, hybridization analysis of virus collected between 1975 and 1980 suggested that the variation among rotavirus strains may have occurred by a process involving both "drift" and "shift" in the sequence of the rotavirus genomic segments.     

Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization

The possibility of an association of mRNA with the cytoskeletal framework (CF) of ascidian (Styela plicata) follicle cells was examined in this study. The approach was to extract the follicle cells with Triton X-100 and determine whether mRNA persisted in the insoluble residue by two methods, in situ hybridization with poly(U) and actin DNA probes and the incorporation of radioactive isotopes into RNA. Triton X-100 extraction of follicle cells yielded a filamentous CF containing approximately 70% of the total poly (A) but only 9% of the total lipid, 23% of the total protein, and 28% of the total RNA. In situ hybridization with a poly (U) probe indicated that approximately 70% of the poly (A) was associated with the CF. In situ hybridization with a cloned actin DNA probe indicated that approximately 60% of the actin mRNA was associated with the CF. Autoradiography of detergent- extracted follicle cells, which had been labeled with [3H]uridine or [3H]adenosine, indicated that greater than 90% of the newly synthesized poly (A)+RNA was preserved in the CF. Thus more newly synthesized mRNA than steady-state mRNA may be present in the Triton X-100 insoluble fraction. It is concluded that a significant proportion of the mRNA complement of ascidian follicle cells is associated with the CF.     

Mechanomyography is more sensitive than EMG in detecting age-related sarcopenia

The aim of this work was to investigate the effects of age-related sarcopenia on the time and frequency domain properties of lower extremity muscles’ electromyographic and mechanomyographic activities. Healthy elderly (n=10, 64.5±4.5 yr) and young (n=10, 22.6±2.8 yr) were recruited as participants. Participants’ lean thigh volumes (LTV) and 1 RM (one repetition maximum) leg strength of quadriceps and maximum speed knee extension with different load levels (45%, 60% and 75% 1 RM) were recorded. The root mean square (RMS) and the mean frequency (MF) of the surface electromyography (EMG_RMS, EMG_MF) and mechanomyography (MMG_RMS, MMG_MF) signals were collected at vastus lateralis during concentric contraction with different intensity levels. Compared to the young, the elderly had significantly less LTV, absolute and relative maximal force, as well as absolute and relative maximal power (p<.05). EMG_MF of the elderly and the young increased monotonically from 45% to 75% 1 RM testing conditions. While the MMG_RMS of the young increased with testing intensities, the MMG_RMS of the elderly increased only from 45% to 60% but leveled off from 60% to 75% 1 RM testing conditions. The results indicate the declines of muscle mass, force and power production capacity with aging. The observations could be explained by neuromuscular performance and change of MU activation patterns may result from age-related sarcopenia. Aging affected muscle power more than muscle strength, which could be due to fast fiber reduction. This is supported by our observations that the MMG_RMS differences between the young and the elderly across all three intensity level where EMG_RMS was only different at the greatest intensity. We suggest that MMG could be used as an important measurement in studying muscle contraction in age-related sarcopenia.     

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.     

Blotting of RNA onto ion exchange paper allowing subsequent characterization by in situ translation in addition to blot hybridization.

We present the preparation of an ion exchange paper which can be used as a solid carrier in the transfer of RNA from gels. In addition to detection by blot hybridization to specific probes, transferred RNA can be characterized by cell-free translation in situ. Preparation of the paper is simple, inexpensive and reproducible. Examples of applications are shown and possible other applications are discussed.     

Rapid pre-screening is more sensitive in liquid-based cytology than in conventional smears

Rapid pre-screening (RPS) is a useful tool to measure and improve performance in the cytology laboratory. Whether RPS is more or less effective in liquid-based cytology than in conventional smears is unknown. We compared the estimated sensitivity in a laboratory of 11 cytotechnologists which converted from conventional smears to SurePath? (Becton Dickinson, Franklin Lakes, N.J., USA) liquid based cytology. In the 9 months prior to conversion, 23,286 smears were screened compared with 30,610 smears in the 12 months immediately after conversion. The estimated sensitivity of rapid pre-screening for 90 s improved significantly with liquid based cytology for all abnormalities (58.7 vs. 68.7%, p<0.001), atypical squamous cells of undetermined significance+low-grade squamous intra-epithelial lesion (52.6 vs. 63.1%, p<0.001), and high-grade squamous intra-epithelial alone (76.2 vs. 85%, p<0.001). Histologic follow up for 156 cases identified by rapid pre-screening of SurePath slides showed 32 (21%) cases of CIN1 or greater and 18 cases (12%) with CIN3 or worse. We conclude that rapid pre-screening is significantly more sensitive in liquid-based cytology compared with conventional smears, and detects significant lesions that are missed by routine screening.     

Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.     

Heterogeneous expression of glutamine synthetase mRNA in rat liver parenchyma revealed by in situ hybridization and Northern blot analysis of RNA from periportal and perivenous hepatocytes

Using radiolabeled specific cDNA glutamine synthetase mRNA could be detected by in situ hybridization exclusively within those few perivenous hepatocytes which stained immunocytochemically for glutamine synthetase. This localization of glutamine synthetase mRNA was recently reported by Moorman et al. [(1988) J. Histochem. Cytochem. 36, 751-755]. Biotinylated cDNA was not suitable for mRNA detection because of a very high background staining under the conditions of in situ hybridization. Dot blot and Northern blot analysis of RNA isolated from periportal and perivenous subfractions of hepatocytes also demonstrated the exclusive perivenous localization of two hybridizable glutamine synthetase mRNAs of length 2.8 and 1.6 kilobases. These results indicate that the unique heterogeneity of glutamine synthetase in rat liver parenchyma is controlled at the pretranslational level.     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

Preparation of giant RNA from bovine thyroid gland by agarose gel filtration

Giant RNA's were prepared by a rapid gel-filtration technique. DNAase, pronase and thyroid homogenate were successively placed on the column. Proteins and DNA were degraded during filtration. Void volume contained pure giant RNA's. Giant RNAs' yield was 7 times higher with this method than with phenol method.