First-trimester endocervical irrigation: feasibility of obtaining trophoblast cells for prenatal diagnosis.

We sought to determine the feasibility of obtaining trophoblast cells for first-trimester prenatal diagnosis using endocervical irrigation. We studied 20 pregnant patients between 7-10.5 weeks' gestation who presented for elective pregnancy termination. Under ultrasound guidance, a specially designed plastic catheter was advanced to the level of the internal cervical os. Gentle flushing and aspiration was performed with 3 mL of normal saline. The material obtained was fixed and stained. A placental pathologist identified trophoblast cells using light microscopy. In another five cases, we attempted to culture the endocervical washings. Trophoblast cells were identified by microscopy after staining the cultured material with an anti-alpha-hCG-antibody bound stain. In ten of 20 cases (50%), trophoblast material was retrieved on irrigation. Of the five additional cases on which culture was attempted, trophoblast was successfully cultured in one, the results were equivocal in two, and culture was unsuccessful in the other two. Trophoblast cells for prenatal diagnosis can be obtained in a significant percentage of cases by first-trimester endocervical irrigation. The advantages of irrigation include technical simplicity, brief duration (less than 3 minutes), and suitability to first-trimester diagnosis. Further testing is necessary to determine the risks.

The biomechanical significance of herniated lumbar intervertebral disk: a clinical comparison analysis of 22 multiple and 39 single segments in patients with lumbar intervertebral disk herniation.

OBJECTIVE: To analyze the clinical significance of herniated lumbar intervertebral nucleus pulposus (HNP) inside the spinal canal. STUDY DESIGN: This was a comparison study between patients with multiple lumbar disk herniation and those with single lumbar disk herniation receiving conservative treatment. METHODS: Four indices, including computed tomography, plain and dynamic radiology and quantified physical examinations, were compared among 22 consecutive patients with multiple disk protrusions and 37 with single disk protrusion before and after a period of conservative treatment, mainly using spinal manipulation. RESULTS: There were no obvious changes of HNP size, position and volume, even after clinical improvement. However, structural and functional recovery in the group with multiple segments was less satisfactory than that of the group with single segment involvement. CONCLUSIONS: The protruded disk tissue plays an important role in the biomechanical disturbances of the spinal column besides that of an irritating agent to nerve root(s).

Iodometric method for detection of beta-lactamase activity in yeast cells carrying ampicillin resistance gene in chimeric plasmids.

In order to determine whether an ampicillin resistance gene in a chimeric plasmid is active in transformed yeast cells, it is necessary to have a simple and quick assay procedure. We describe here a procedure for achieving this goal using an iodometric color reaction. This method is based on the fact that the ampicillin resistance gene product, beta-lactamase, can hydrolyze penicillin G and release a reducing product, which can be visualized by the discoloration of a dark blue iodine-starch complex. We have improved this method so that the assay can be carried out on agar plate and in liquid culture. It permits the detection of the beta-lactamase enzyme activity in yeast liquid culture at a concentration as low as 1 X 10(5) cells/ml within 12 h. This method is especially useful for certain yeast transformation systems, such as industrial yeast cultures, where the transformants can be selected only by drug resistance.

Protruded lumbar intervertebral nucleus pulposus in a 12-year-old girl who recovered after nonsurgical treatment: a follow-up case report.

OBJECTIVE: To discuss the nonsurgical treatment of a lumbar intervertebral disc protrusion with obvious neurological irritation in a juvenile patient. CLINICAL FEATURES: A 12-yr-old girl suffered from low back and right leg pain that came on after a lengthy walk. There was a limp on the right, with pain also radiating to the right leg. A diagnosis of lumbar disc herniation was made after advanced imaging methods were applied. INTERVENTION AND OUTCOME: The girl underwent special spinal rotational manipulation, epidural injection and, later on, physical exercises. She did not respond to the manipulation as adult patients with similar signs do; in her case, response did not occur until nearly 1 month of hospitalization. A follow-up study with computed tomography, plain and dynamic radiology and thermography found that the protruded nucleus pulposus was not changed in size and position and the functional recovery of her lumbar spine was far from fulfilled until 8 months after being dismissed from the hospital. CONCLUSION: Juvenile patients suffering from lumbar disc protrusion are rare and seldom undergo conservative treatments, especially when obvious neurological irritation occurs. This case shows the difficulties in managing the juvenile patient with conservative care. Doctors or specialists should always be cautious about manipulating juvenile patients with too much force.

The persistence of maternal inheritance in Chlamydomonas despite hypomethylation of chloroplast DNA induced by inhibitors.

We have used inhibitors of methylation to evaluate the proposal that the extent of methylation of chloroplast DNA ( cpDNA ) of the mating type-plus (mt+) parent occurring during gametogenesis in wild-type Chlamydomonas renhardtii is directly correlated with the uniparental transmission of chloroplast genes by this parent [ Sager , R., Grabowy , C. & Sano , H. (1981) Cell 24, 41-47]. As detected by high-pressure liquid chromatography, the methylation of cpDNA was at its lowest level in the vegetative stage; the mt+ cells had a deoxycytidine methylation index (the percentage of deoxycytidine methylated) of 0.5, while the mating type-minus (mt-) index was lower by at least a factor of 3. This basal level of cpDNA methylation increased more than 20-fold after gametogenesis to give a methylation index of 12.1 and 4.3 for mt+ and mt- gametes, respectively. Another striking increase was detected at the 7-hr-zygote stage, resulting in the methylation of nearly half of the total deoxycytidine residues. The extent of zygotic cpDNA methylation was shown to be dependent on the preexisting methylation level of both parental gametic cpDNAs . L-Ethionine and 5-azacytidine effectively inhibited cpDNA methylation during gametogenesis and ensuing zygotic development as shown by both Hpa II/Msp I digestion patterns and HPLC. The transmission of chloroplast genes was analyzed concomitantly with the inhibitor studies. The two inhibitors produced different patterns of inhibition of methylation in mt- and mt+ cells at a given developmental stage. Our overall results demonstrate that the extent of mating type-specific and gamete-specific methylation during gametogenesis is not correlated with the frequency of maternal transmission of chloroplast genes.

Characterization of deoxycytidylate methyltransferase in Xanthomonas oryzae infected with bacteriophage Xp12.

Three methods, chromatographic, spectrophotometric and tritium-release assay, were used and compared for the assay of deoxycytidylate methyltransferase. All three methods can be used for assay of this enzyme but the tritium-release assay appears to be the most simple and convenient. With the help of this assay the deoxycytidylate methyltransferase has been isolated and purified from sonically disrupted cells of Xp12-infected Xanthomonas oryzae. Using a procedure that involves fractionation with streptomycin sulfate and ammonium sulfate, filtration through Sephadex G-100 and chromatography on DEAE-cellulose, a 214-fold increase in specific activity was obtained. The enzyme displays a narrow pH optimum at 6.0 Among the buffers tested, 6-morpholinoethane sulfonate with the addition of Mg2 is the best. The enzyme can utilize dCMP as a substrate. The enzyme can also convert tetrahydrofolic acid into dihydrofolic acid. The Km value for dCMP is 31.3 micrometer and the Km value for tetrahydrofolic acid is 71.4 micrometer. There is no absolute requirement of ions for the activity of the enzyme; however, the presence of ions causes stimulating or inhibiting effects on enzyme activity that are dependent on the variety and concentration of ions used.

Chloroplast segregation in somatic hybrids of Nicotiana plumbaginifolia and N. sylvestris having different ratios of parental nuclear genomes.

Fusion of mesophyll protoplasts of haploid Nicotiana plumbaginifolia (P) and N. sylvestris (S) resulted in the production of somatic hybrid plants of various ploidy levels. Analysis of the restriction fragment patterns of chloroplast DNA from 118 plants belonging to genome constitutions PS, PPS, PSS, and PPSS revealed that two had a pattern corresponding to a mixture of parental DNA while all the others had the pattern of either N. plumbaginifolia or N. sylvestris. In the latter case, the ratio of the two parental types fits 1∶1 in all the four genome constitutions studied. Since the protoplasts used in the fusion experiment were physiologically similar and the hybrid cells were not deliberately selected, these results suggest that chloroplast segregation in the somatic hybrids is independent of the chloroplast input of the fusion partners and the nuclear background of the fusion products.

Characterization of phage-Xp10-coded RNA polymerase.

A bacteriophage-coded RNA polymerase was isolated from bacteriophage-Xp10-infected Xanthomonas campestris pv. oryzae. The enzyme was purified to homogeneity through precipitation by poly(ethylene glycol) and chromatography on DEAE-cellulose, heparin--Sepharose 4B and blue-dextran--Sepharose 4B. It is composed of a single polypeptide of Mr96,000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA, host bacterium DNA and poly[d(A-T)] as templates. The optimal concentration of MgCl2 is 16 mM. The optimal temperature and pH are 37 degrees C and 8.0, respectively. The Km of ATP is 26 microM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xp10 RNA polymerase activity was retained. The enzyme activity was inhibited by KCl, spermidine, actinomycin D, heparin, blue dextran and ethidium bromide; it was resistant to rifampicin and streptovaricin. N-Ethylmaleimide did not affect the enzyme activity. The transcription site and product of Xp10 RNA polymerase upon Xp10 DNA were analyzed by DNA/RNA hybridization and polyacrylamide-agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xp10 genome and produce two RNA bands.

Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia.

Chloroplast DNA isolated from wild-type Nicotiana plumbaginifolia and 12 maternally inherited streptomycin-resistant mutants was digested with various restriction enzymes and the resultant patterns were compared. No gross structural alterations of the chloroplast genome were detected in any mutants; however, variant patterns owing to the gain or loss of a restriction site were found in three mutants, SR1007, SR1019, and SR1022. The variant patterns in SR1019 and SR1022 are identical and are the results of mutation in the psbG gene coding for a chloroplast membrane protein G, and that in SR1007 is due to mutation in the 16S rRNA gene. Inheritance of the variant patterns in mutants SR1007 and SR1019 was studied. The results showed that the variant patterns and streptomycin resistance were co-transmitted in reciprocal crosses.

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure.

The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.

cDNA cloning and characterization of a plant protein that may be associated with the harpinPSS-mediated hypersensitive response.

Hypersensitive response-assisting protein (HRAP) is a novel plant protein that can intensify the harpinPSS-mediated hypersensitive response (HR) in harpinPSS-insensitive plants, such as the vegetative stage of sweet pepper. In this report, we identified a HRAP cDNA clone from sweet pepper (Capsicum annuum cv. ECW). The sequence of this cDNA clone showed no appreciable similarity to any other known sequences. However, it contained three positively charged regions, a typical signal peptide and a cAMP-dependent phosphorylation site. The hrap mRNA accumulated preferentially during the incompatible interaction of sweet pepper leaves with a pathogenic bacterium, Pseudomonas syringae pv. syringae. When the hrap gene transcription level was high, the sweet pepper leaves readily expressed the harpinPSS-mediated HR. The hrap gene transcription level in sweet pepper was also higher during the reproductive stage than during the vegetative stage. The HRAP distribution in an individual plant and different plant species was investigated. We found that all the organs of sweet pepper, except fruit, could express two different forms of HRAP. Moreover, the hrap gene was presented in many plant species including tobacco, Arabidopsis, and rice. In conclusion, our results suggest that the hrap gene is widely distributed throughout the plant world and its transcription level correlates with plant sensitivity to harpinPSS. The interaction between HRAP and harpinPSS reveals a novel way to interpret the interaction mechanism between plants and bacterial pathogens.

Plasmids in diatom species.

We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements.

Determination and Distribution of cry-Type Genes of Bacillus thuringiensis Isolates from Taiwan.

Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, we were able to screen the cry-type genes of 225 Bacillus thuringiensis soil isolates from Taiwan without much cost in time or labor. Some combinations of cry genes (the cry-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the B. thuringiensis soil isolates from Taiwan. The cry genes included cryIA(a), cryIA(b), cryIA(c), cryIC, cryID, and cryIV. Interestingly, 501 B. thuringiensis isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of cry-type genes were distinct in all isolation areas. The distribution of cry-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect cryIC-type genes, we identified two distinct cryIC footprints from some of our isolates, indicating that these isolates may contain novel cryIC-type genes. B. thuringiensis isolates containing cryIA(a)-, cryIA(b)-, and cryIA(c)-type genes exhibited much greater activity against Plutella xylostella than did other isolates, indicating that multiple cry-type genes may be used as markers for the prediction of insecticidal activities.

Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host.

It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host. Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells. DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared. Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific. Only one complement of viral DNA was integrated per host chromosome. To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed. Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences. The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA.

The lysogenic cycle of the filamentous phage Cflt from Xanthomonas campestris pv. citri.

A phage, Cflt, forming turbid plaques, was isolated from Xanthomonas campestris pv. citri. After infection, infected sensitive cells become immune to Cflt and produce very few phages. These properties were genetically rather stable. The phage was purified and shown to be filamentous with a size of 1157 +/- 73 nm. The genome size is about 7.62 kb. The phage does not affect the growth of host bacteria. Under natural cultivation conditions Cflt-lysogenized cells could be induced spontaneously to give high phage yields, or cured to give phage-free cells. The integration of Cflt DNA into host DNA was proved by Southern blot hybridization. The lysogenic phage was genetically stable in log phase cells and persisted in stationary phase cells through many cell generations in the absence of extracellular phage reinfection.

Characterization of two circular plasmids from the marine diatom Cylindrotheca fusiformis: plasmids hybridize to chloroplast and nuclear DNA.

This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.

Controlled fed-batch fermentation of recombinant Saccharomyces cerevisiae to produce hepatitis B surface antigen.

We have performed controlled fed-batch fermentation experiments to compare the production level of hepatitis B surface antigen (HBsAg) by recombinant yeast Saccharomyces cerevisiae strains (YNN27/pYBH-1, YNN27/ p2micro-S11, YNN27/pDCB-S2) containing plasmid vector with alcohol dehydrogenase (ADH1), acid phosphatase (PHO5), and glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, respectively. Yeast cell concentrations of 15-35 g dry cell weight/L were obtained. By limiting phosphorous concentration, HBsAg expression level for the YNN27/p2micro-S11 strain with inducible PHO5 promoter reached 0.2-0.3 mg/L. By controlling nutrient addition rate and dissolved oxygen concentration, HBsAg concentrations of 3-10 mg/L were achieved in 60-70 h fermentation using the YNN27/pDCB-S2 strain with the constitutive GPD promoter.