Regulation of small RNAs in Escherichia coli. Alteration in the intracellular concentrations of small RNAs during amino acid and energy starvation.

The accumulation of low molecular weight RNAs in Escherichia coli cells following amino acid or energy source starvation was examined using two-dimensional polyacrylamide gel electrophoresis. 32P-labeled small RNA prepared from serine- or isoleucine-starved stringent strain (relA+) cells was shown to display gel patterns that were grossly different from that of unstarved cells. It appears that the deprivation of serine or isoleucine has little or no inhibitory effect on the accumulation of transfer RNA cognate to the deprived amino acid. This is demonstrated by a relative increase in the concentrations of small RNAs that can be charged with serine or isoleucine following starvation of these amino acids. However, small RNAs labeled during starvation of phenylalanine or energy source showed gel patterns similar to that of control cells. This suggested a heterogenous response in the accumulation of some low molecular weight RNAs, presumably transfer RNAs, following starvation of different amino acids.

Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes.

Unusual guanosine nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp, also known as MSI) and guanosine 5'-diphosphate 3'-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation. We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways. In both cases, ppGP (MSII) is a stronger inhibitor than ppGpp (MSI). On the other hand, these two nucleotides exhibited opposite effects on the activity of phosphoenolpyruvate carboxylase, the enzyme that utilizes phosphoenolpyruvate. At their respective physiological concentrations, the activity of phosphoenolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.

Detection of human papillomavirus types 16 and 18 mRNA in peripheral blood of advanced cervical cancer patients and its association with prognosis.

PURPOSE: To evaluate the feasibility of detecting human papillomavirus E6 (HPVE6) gene mRNA in the peripheral blood of patients with locally advanced cervical cancer, and the relationship of the circulating HPV viral-specific mRNA with clinicopathologic factors and prognosis of locally advanced cervical cancer. PATIENTS AND METHODS: The presence of types 16 and 18 HPVE6 gene mRNA was determined by reverse transcription followed by nested polymerase chain reaction. Thirty-five patients with locally advanced cervical cancer who were positive for HPV type 16 or 18 DNA were included in the study. All patients received external-beam radiation therapy followed by intracavitary brachytherapy. RESULTS: Eighteen (51.4%) of 35 HPV DNA-positive cervical cancer patients had HPV-specific mRNA in their peripheral blood cells, compared with none of 17 HPV DNA-negative cervical cancer patients and none of 12 control volunteers. The presence of HPVE6 gene mRNA in peripheral blood was associated with bulky tumor volume (> 4 cm) and pelvic lymph node metastasis (tumor volume, P = .03; lymph node status, P = .03). After a median follow-up of 22 months, patients who were positive for peripheral-blood HPVE6 gene mRNA had a significantly higher risk of recurrence than those who were negative (10 of 18 v three of 17, P = .02; mean recurrent time, 20.7 months v 12.6 months, P = .02). There was also a statistically significant association of peripheral-blood HPVE6 gene mRNA positivity with distant metastasis (eight of 18 vone of 17; P = .01). CONCLUSION: Results of this study seem to suggest that the presence of HPVE6 gene mRNA in peripheral blood may provide an early marker that identifies patients who are at risk for metastasis.

Detection of human papillomavirus mRNA and cervical cancer cells in peripheral blood of cervical cancer patients with metastasis.

PURPOSE: To determine the presence of cervical cancer cells in circulating peripheral blood of stage IVb cervical cancer patients with metastasis to distant organs. PATIENTS AND METHODS: Cervical cancer tissue from 15 stage IVb cervical cancer patients with metastasis were analyzed for the presence of human papillomavirus (HPV) type 16 DNA by nested polymerase chain reaction (PCR). The presence of transcriptional products of the HPV type 16 E6-transforming gene in the peripheral blood of the same 15 cancer patients was analyzed by reverse transcription and PCR. Cervical tissues and peripheral-blood specimens from 12 normal healthy individuals served as controls. RESULTS: Thirteen of 15 (86.7%) cervical cancer tissues from same number of patients were found to contain HPV type 16 DNA. Peripheral-blood specimens from 12 of 13 (92.3%) cervical HPV DNA-positive patients were found to contain HPV-specific mRNA detectable by reverse transcription (RT) and PCR. Cervical tissues from all 12 normal controls were HPV-free. None of the peripheral-blood specimens from two cervical HPV-negative cancer patients and 12 normal controls contained detectable amounts of mRNA of HPV type 16 E6-transforming gene. CONCLUSION: The most likely source of the HPV-specific mRNA detected in the peripheral blood of cervical cancer patients with metastasis is the cervical cancer cells derived from or shed from the cervix. The presence of HPV E6 mRNAs in peripheral blood may be a sensitive indicator of circulating cervical cancer cells. If PCR positivity is proven to be able to predict disease progression reliably, these findings may have clinical applications in the treatment of cervical and many other cancers.

Differential expression of telomerase activity in human cervical cancer and cervical intraepithelial neoplasia lesions.

PURPOSE: Telomeres are tandem arrays of repeated DNA sequences located at the ends of eukaryotic chromosomes, and are synthesized by the enzyme telomerase. Loss of telomeric DNA may play an important role in the development of human cancers. However, very little is known about the status of telomerase during human cervical cancer development. PATIENTS AND METHODS: Telomerase activity was measured by telomere repeat amplification protocol (TRAP) assay in 24 cervical cancers, one carcinoma in situ (CIS), and 20 cervical intraepithelial neoplasia (CIN) lesions. Adjacent nontumor cervical tissue from the same 24 cervical cancer patients and normal cervical tissues from 11 control individuals also were examined for the presence of telomerase activity. RESULTS: Twenty two of the 24 (91.7%) cervical cancer specimens and the single CIS tissue were strongly positive for telomerase activity. Relatively weak but distinctive telomerase activity also was detectable in one of four CIN-I (25%), two of eight CIN-II (25%), and two of eight CIN-III (25%), respectively. However, telomerase activity was not found in the 24 corresponding nontumor cervical tissues from the same cervical cancer patients and the 11 normal cervical tissues from control individuals. CONCLUSION: The majority of cervical cancers contain strong telomerase activity. Significant proportions of noncancerous CIN tissues also contain telomerase activity, although weaker than that in cervical cancer. It seems that there is a progressive increase of telomerase activity in association with an increased degree of cervical malignancy. These results seem to suggest that the expression of telomerase may play a crucial role in cervical cancer carcinogenesis.

Differential expression of interleukin-1 beta and interleukin-6 in human fetal serum and meconium-stained amniotic fluid.

The study was designed to investigate the expression of the inflammatory cytokines interleukin-1 beta and interleukin-6 in meconium-stained amniotic fluid and in fetal cord serum. Amniotic fluid and fetal cord serum specimens were collected from 10 and 9 women with meconium-stained and clear amniotic fluid, respectively, during Caesarean operation at labor The mean concentrations of interleukin-1 beta found in clear and meconium-stained amniotic fluid were 10.0 and 54.5 pg/ml, respectively, and the difference was not statistically significant. On the other hand, the concentrations of interleukin-6 in meconium-stained amniotic fluid (774 pg/ml) was significantly higher than that found in clear amniotic fluid (149 pg/ml) (P = 0.0036). The differences of levels of both interleukin-1 beta and interleukin-6 in fetal cord serum specimens were not significant between neonates born to mothers with either clear or meconium-stained amniotic fluid (P = 0.8702 and 0.2987, respectively). The results of this study suggest that the production of at least one of the inflammatory cytokines, interleukin-6, is associated with the meconium found in amniotic fluid.

Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening.

In an attempt to seek out new factors that are related to colorectal carcinogenesis at the molecular level, subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues was performed. Subsequent screenings of the cDNA libraries, constructed from normal mucosal tissues, using the "subtractive probes" generated a total of 46 clones that were expressed in normal mucosa but were either expressed at a significantly reduced level or not expressed at all in cancer tissues. Partial nucleotide sequences of all of these cDNA clones were determined, and sequence homology analyses were performed with the Genbank database. Of the 46 cDNA samples, 44 contained substantial sequence homologies with 32 immunoglobulin gene fragments, a helix-loop-helix basic phosphoprotein gene, an acidic ribosomal phosphoprotein P2 gene, a BLR1 gene for Burkitt's lymphoma receptor 1 gene, D5S419 DNA segment containing (C-A) repeats, a glucokinase (GCK) gene, a Na+, K+-ATPase alpha-subunit gene, a histocompatibility system HLA-DR heavy-chain gene, a dystrophic gene, a mucin (MUC2) gene, a mu-glutathione S-transferase gene, a Menkes disease protein gene, and a 40-kDa keratin intermediate filament precursor gene. The remaining two cDNA clones (now registered under GenBank accession numbers U17714 and U20428) showed few (less than 60%) sequence homologies with any known sequences in the GenBank database and, therefore, may represent novel genes whose expression was down-regulated in human colorectal carcinomas. The possible clinical significance of these findings and the involvement of these two genes in the carcinogenesis of colorectal as well as other cancers are being investigated.

Hepatitis C virus in peripheral blood mononuclear cells.

RNA extracted from plasma and peripheral blood mononuclear cells of patients with chronic hepatitis C were used as the template for reverse transcription followed by double in vitro enzymatic amplification with nested primers. Hepatitis C virus was detected in 14 of 15 (93.3%) plasma specimens and in 8 of 15 (53.3%) peripheral blood mononuclear cell specimens obtained from patients with chronic hepatitis C and abnormal liver functions. The results suggest that hepatitis C virus could be found frequently in peripheral blood mononuclear cells of patients with chronic hepatitis C. Whether the presence of hepatitis C virus in peripheral blood mononuclear cells plays any role in the pathogenesis of diseases associated with hepatitis C virus infection remains to be determined.

Identification of human papillomavirus DNA sequences in peripheral blood mononuclear cells.

Polymerase chain reaction (PCR) was used to amplify and identify the presence of the DNA of human papillomavirus (HPV) types 6, 11, 16, and 18 in peripheral blood mononuclear cells (PBMCs) of women with and without urogenital HPV infections. HPV DNA of various types was found in PBMCs of 13 of 25 (52.0%) patients with urogenital HPV infections and in none of the 19 control subjects who are free of urogenital HPV infections. The presence of HPV DNA in PBMCs may impair the immunologic functions of the lymphocytes and play a role in the epidemiology of HPV infections and the pathogenesis of HPV-induced diseases.

Serum hepatitis B virus DNA in hepatitis B virus seropositive and seronegative patients with normal liver function.

The presence of hepatitis type B virus (HBV) DNA in serum specimens from 926 apparently healthy people with normal liver functions was determined by polymerase chain reaction; 41.2% of people with positive results for HBV surface antigen (HBsAg) (94 of 228) and 95.2% of people with positive results for HBV e antigen (HBeAg) (60 of 63) were found to have positive results for serum HBV DNA. On the other hand, serum HBV DNA was found in 11.0% (77 of 698) of HBsAg-negative people and in 13% (69 of 530) of those who had positive results for serum antibodies directed against HBsAg. The results seem to suggest that HBV DNA can be found in a significant portion of apparently healthy people with normal liver function who are either seronegative for HBsAg or seropositive for antibodies directed against HBsAg.

Presence of cells of fetal origin in maternal circulation of pregnant women.

Fetal cells can be identified by using the polymerase chain reaction to test for the presence of human Y-chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood of women who bear a male fetus. Thirty-one pregnant women were studied in the first trimester to determine when fetal cells become detectable in the maternal circulation. Among the 19 women whose peripheral blood samples were positive for Y-chromosome-specific DNA sequences, the presence of fetal cells was quite case-variable from the 6th to 12th gestational weeks. Twenty-eight women who had given birth to their first male babies were studied postpartum to determine when fetal cells disappear from the maternal circulation. Fetal cells can still be detected in maternal blood 10 months postpartum in some cases. These results suggest that identification of fetal cells in the maternal circulation is possible. Nevertheless, interpretation of fetal cells in maternal circulation should be handled very carefully with respect to when these fetal cells first became detectable and potential interference from previous pregnancies.

Perinatal transmission of human papillomavirus in infants: relationship between infection rate and mode of delivery.

OBJECTIVE: To determine the transmission rate of human papillomavirus (HPV) in newborn infants of HPV-positive women and to assess the relationship between perinatal HPV transmission and mode of delivery. METHODS: Three hundred one pregnant women were selected: vaginal delivery (n = 160) or cesarean delivery (n = 141). We assessed the presence of the HPV types 16 and 18 DNA sequences in buccal and genital swabs of neonates born to HPV-positive mothers, using the polymerase chain reaction. RESULTS: The overall frequency of HPV 16/18 infection among the pregnant women was 22.6% (68/301). At birth, the overall frequency of HPV transmission from HPV 16/18-positive mothers to newborns was 39.7% (27/68). A significantly higher rate of HPV 16/18 infection was found at birth when infants were delivered vaginally than when infants were delivered by cesarean (18/35 or 51.4% versus 9/33 or 27.3%, P = .042). However, there was no significant difference in the incidence of perinatal HPV infection between the HPV types 16 and 18 in either vaginal delivery group or in the cesarean delivery group (all P > .100). No significant difference was found between the buccal and genital sites (27/68 versus 21/68, P = .234) or between male and female infants overall (12/36 versus 15/32, P = .255). CONCLUSION: The findings suggest that neonates are at higher risk for exposure to HPV after vaginal delivery than after cesarean delivery.

Human papillomavirus deoxyribonucleic acid and ribonucleic acid in seminal plasma and sperm cells.

OBJECTIVE: To investigate the possible presence and expression of human papillomavirus viruses (HPV) in human plasma and sperm cells. DESIGN: Controlled clinical study. SETTING: A major medical center affiliated with a medical college. PATIENTS: Twenty-four randomly selected patients who attended Fertility Clinics at the Chang Gung Memorial Hospital. INTERVENTIONS: Specimens of semen were collected from volunteered patients MAIN OUTCOME MEASURE: The presence of HPV types 16 and 18 DNA and RNA sequences were examined by polymerase chain reaction. RESULTS: Human papillomavirus type 16 E6 and E7 DNA and RNA sequences were found in two and zero seminal plasma specimens, respectively, and in six and two sperm cells specimens, respectively. Deoxyribonucleic acid and RNA sequences of HPV type 18 were found in eight and two seminal specimens and in 11 and 5 sperm cells specimens, respectively. CONCLUSION: These results seem to suggest that HPV cannot only infect human sperm cells, certain HPV genes are expressed actively in infected sperm cells. The virus-infected sperm cells conceivably can behave as vectors or carriers for the transmission of HPV, to sexual partner during sexual contact, to fetuses through fertilized eggs, or both.

Preferential retention of the E6 and E7 regions of the human papilloma virus type 18 genome by human sperm cells.

OBJECTIVE: To determine if sperm cells differentially take up or retain different regions of human papilloma virus (HPV) type 18 genome. DESIGN: A descriptive clinical study. SETTING: A major medical center affiliated with a medical college. PATIENTS: Twenty-three randomly selected patients who attended Fertility Clinics at the Chang Gung Memorial Hospital. Semen specimens were obtained from volunteered patients who attended fertility clinics at the Chang Gung Memorial Hospital. MAIN OUTCOME MEASURE: The presence of various regions of HPV type 18 genome in sperm cells was determined by polymerase chain reaction. RESULTS: Among 23 sperm specimens that were positive for HPV type 18 DNA by polymerase chain reaction, the upstream regulatory region, E6, E7, E1, and L1 regions or open region frames were detected in 4 (17%), 7 (30%), 19 (83%), 5 (22%), and 1 (4%) specimens, respectively. The differential display of the presence of various regions of the HPV type 18 genome was not the result of different amplification and detection efficiencies of these DNA fragments. CONCLUSION: These results suggest that the oncogenic portion of HPV DNA is present in spermatozoa. Furthermore, the E6 and E7 regions of the viral genome preferentially were taken up and/or retained by the human sperm cells.

Endocervical chlamydial deoxyribonucleic acid in infertile women.

The presence of chlamydial deoxyribonucleic acid (DNA) was evaluated by DNA hybridization in endocervical cells of infertile and normal fertile women. Chlamydial DNA was detected in 49 of 186 (26.3%) infertile patients, which is significantly more common than in fertile control individuals (12.5%, or 8 of 64 individuals). Among infertile patients, 49.3% (33 of 67) of those with tubal factors as cause of infertility and 13.4% (16 of 119) of those with nontubal factors were found to contain chlamydial DNA in their endocervical cells. The results show that chlamydial DNA could be found significantly more frequently in endocervical cells of infertile patients with tubal factor than those without tubal factors or in normal controls.

The effect of human papillomavirus infection on sperm cell motility.

OBJECTIVE: To investigate the presence of human papillomavirus (HPV) in human sperm cells and to evaluate potential effects of HPV on the sperm functions. DESIGN: A descriptive clinical study. PATIENT(S): Specimens of semen were collected from 24 randomly selected patients who attended the fertility clinics at Chang Gung Memorial Hospital. MAIN OUTCOME MEASURE(S): The presence of HPV DNA and RNA were examined by polymerase chain reaction. Semen quality and sperm cell function were analyzed by computer-aided autoanalyzer. RESULT(S): Human papillomavirus type 16 DNA and RNA were found in 6 (25%) and 2 (8%) of the sperm cells specimens, respectively. Human papillomavirus type 18 DNA and RNA were present in 11 (46%) and 5 (21%) of the same sperm cells specimens, respectively. Incidence of asthenozoospermia among patients infected with either HPV was significantly higher than in those without HPV in their sperm cells (75% versus 8%). Although performance of curvilinear velocity, straight-line velocity, and mean amplitude of lateral head displacement was significantly lower in HPV-infected specimens, the differences of linearity, beat cross frequency, and straightness were not statistically significant. CONCLUSION(S): These results suggest that human papillomavirus can be found in human sperm cells and that certain HPV-specific genes are actively transcribed. Sperm motility parameters seem to be affected by the presence of HPV in the sperm cells, and also the incidence of asthenozoospermia may be associated with HPV infection.

Amplification of viral RNA for the detection of dengue types 1 and 2 virus.

In vitro DNA amplification by means of the polymerase chain reaction (PCR) was used to amplify dengue types 1 and 2 viral genomes in cultured cells and in the serum of persons infected with dengue virus. Results of the present investigation suggest that the PCR method is type-specific in detecting dengue virus and has a detection sensitivity of less than 100 plaque-forming units (pfu) for both serotypes of the virus. The PCR method may be useful for detecting and typing dengue virus in clinical and epidemiological specimens.

Hepatitis B virus DNA in cervicovaginal cells.

Hepatitis B virus DNA sequences were detected in seven (12.1%) of 58 cervicovaginal cell specimens that were obtained from pregnant women by polymerase DNA amplification assay. The presence of hepatitis B virus DNA in these cells raises the possibility that infected cervicovaginal cells may be a source through which hepatitis B virus can be transmitted from infected mothers to their newborns and between heterosexual partners.