First Report of Podosphaera xanthii Race 1W Causing Powdery Mildew of Watermelon in California.

In September and October 2012, powdery mildew was detected on watermelon (Citrullus lanatus var. lanatus) plants of various breeding lines growing in field plots in Davis, California. Plants had partially necrotic leaves, yellowing to brown in color, with white surface mycelium and faint sporulation. No teleomorph was observed. Infected leaves were collected for examination and a spore suspension of the field isolate was made in water with 0.01% Tween 20 to spray inoculate watermelon seedlings of cultivar Dixie Lee with two true leaves. Plants were incubated in a growth chamber (22 to 26°C, 12-h photoperiod) for approximately 10 days, until sporulation was apparent. Microscopic observation of conidial chains showed that they had clearly crenate edges indicative of Podosphaera xanthii (4). To confirm the identity of the pathogen, we used Podosphaera-specific primers PFITS-F (5'-CCAACTCGTGCTGAGTGT-3') and PF5.8-R (5'-TGTTGGTTTCTTTTCCTCCG-3') to amplify and sequence the internal transcribed spacer regions of the nuclear rDNA. The 326-bp sequence had 98% homology to the GenBank sequence (accessions JQ340082.1 and AB774158.1) for P. xanthii. Infected 'Dixie Lee' leaves were used to make a spore suspension (approximately 5 × 104 conidia/ml) as described above to inoculate watermelon, melon, and squash seedlings (2 to 3 plants per cultivar) in a greenhouse. It caused severe symptoms on all watermelon plants cv. Charleston 76, P8, and Sugar Baby in the form of a powdery mildew with surface mycelium and chains of conidia, with leaves becoming gradually more necrotic and eventually dying, with the appearance of a melting down. Non-inoculated plants did not develop symptoms. The isolate also infected all squash plants 'Zucchini Elite' and melon powdery mildew differentials Iran H and 'Védrantais.' On these plants, the pathogen produced a powdery mildew (white surface mycelium with sporulation) but did not cause extensive necrosis. All other melon powdery mildew differentials ('PMR5,' 'PMR45,' WMR29, MR1, PI 124112, and PI 313970) did not develop any powdery mildew. A follow-up test in a growth chamber (22 to 26°C, 12-h photoperiod) with the same set of species and cultivars gave the same results. Based on these results, we conclude that this isolate belongs to race 1W (1,2). The presence of race 1W could have implications in disease management for this crop in the Central Valley of California as most cultivars are not resistant to it and the disease has been shown to cause severe damage in other states (1,3). References: (1) A. R. Davis et al. J. Am. Soc. Hort. Sci. 132:790, 2007. (2) J. D. McCreight. Amer. Soc. Hort. Sci. 131:59, 2006. (3) A. Y. Tetteh et al. Crop Sci. 50:933, 2010. (4) T. A. Zitter. Page 28 in: Compendium of Cucurbit Diseases, The American Phytopathological Society, St. Paul, MN, 1996.

Phylogeny of the families Pyuridae and Styelidae (Stolidobranchiata, Ascidiacea) inferred from mitochondrial and nuclear DNA sequences.

The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style.

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

Residual symptomatic superior oblique palsy.

PURPOSE: To determine the outcome of patients treated for residual symptomatic hyperdeviations, in a tertiary referral centre, following a previous weakening procedure of the ipsilateral Inferior Oblique (IO) muscle in Superior Oblique (SO) palsy. METHODS: A retrospective review of 37 patients seen over 6 years at one institution who had remained symptomatic from a SO palsy despite having had an initial weakening procedure to their ipsilateral IO (myectomy or recession). Median age was 19 years (range 3 to 56 years). Information recorded included pre- and postoperative deviation and ocular motility findings, preoperative symptoms, findings at the time of surgery, and outcome. RESULTS: Nine patients underwent repeat weakening surgery (disinsertion) on the ipsilateral IO only. Thirteen patients underwent strengthening surgery on the ipsilateral SO only. Nine patients had surgery on both the ipsilateral IO and SO. Six patients had surgery on the ipsilateral IO with either horizontal or vertical rectus surgery. Nine (24%) patients remained symptomatic after their initial procedure and are regarded as initial failures. Four of these patients had masked bilateral IO weakness. Five patients required additional surgery. At final outcome, 84% were discharged with resolution of their symptoms. CONCLUSIONS: In the light of these findings we suggest an approach for the management of these patients. This should always include exploring a previously operated ipsilateral IO. Despite this, patients should be warned that they have a 1 in 4 chance of needing further surgery to achieve adequate ocular motility.

Demographic feedback between clonal growth and fragmentation in an invasive seaweed.

Many abundant plants, invertebrates, and seaweed are clonal, and this allows the formation of high-density aggregations, foraging, and the placement of modules into new space, and rapid rates of expansion. For these species, population density and rates of expansion are functions of recruitment of asexual modules and post-recruitment vegetative growth and survivorship. In this study, we provide the first experimental test of the relative importance of these two processes in determining the abundance of a clonal seaweed using Caulerpa taxifolia, an invasive green alga that spreads rapidly and reaches very high abundance. We asked two main questions: What is the relative importance to abundance (biomass) of vegetative stolon growth and fragment recruitment during expansion of established patches? Does greater fragment recruitment result in greater abundance in established patches? Vegetative growth of stolons underpinned patch expansion. Plots with stolons growing into them always had a greater abundance than plots where stolons were removed, even when fragment recruitment was increased. Greater recruitment only resulted in greater abundance when stolons were absent, a situation analogous to the establishment of new populations. Although post-recruitment processes were more important in determining abundance during patch expansion, there was greater ambient fragment recruitment when stolons were present compared to when they were absent, and as the abundance of C. taxifolia increased, demonstrating an important feedback between stolon growth, abundance, and fragment recruitment. In established patches, greater fragment recruitment over six months (six levels ranging from 0 to 480 recruits x m(-2) x mo(-1)) had no effect on biomass. Our experiments demonstrate that the rapid expansion and high abundance of invasive C. taxifolia are underpinned by post-recruitment vegetative growth and, during expansion, by a feedback between vegetative growth and asexual fragmentation.

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences.

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. The key membrane-associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22-phox and gp91-phox. Currently, only the human, porcine, murine, and rattus p22-phox and the human, porcine, and murine gp91-phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22-phox and gp91-phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91-phox and p22-phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis.

Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs.

Neutrophils play an essential role in bovine cellular host defense, and compromised leukocyte function has been linked to the development of respiratory and mucosal infections. During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. Two of the essential cytosolic components of the NADPH oxidase are p47-phox and p67-phox. Currently, only the human and murine homologs of these proteins have been sequenced. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and express bovine p47-phox and p67-phox. Using polymerase chain reaction (PCR) cloning techniques and a bovine bone marrow cDNA library, we have cloned both of these bovine NADPH oxidase cytosolic components. Comparison of the bovine sequences with those of the human and murine homologs showed that they were highly conserved, but also revealed important information regarding key structural features of p47-phox and p67-phox, including location of putative phosphorylation sites. Functional expression of bovine p47-phox and p67-phox showed that these proteins could substitute for the human proteins in reconstituting NADPH oxidase activity in a cell-free assay system, again demonstrating the high degree of conservation between human and bovine homologs. This study greatly contributes to our understanding of the potential structural/functional regions of p47-phox and p67-phox as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases.

Current potential of antisense oligonucleotides as therapeutic drugs.

Antisense oligonucleotides have great potential as rationally designed therapeutic drugs by taking advantage of the basic Watson-Crick base pairing of nucleic acids. Such oligonucleotides may block synthesis of a specific protein, such a c-myb, and prevent smooth muscle proliferation involved in restenosis. Although promising, the technology has some major hurdles to overcome before fruition. These include obtaining a stable backbone that can enter cells readily, overcoming problems of chirality, and solving the problems of delivery and metabolism. Although there are many reports of successful experiments using antisense oligonucleotides, one must always keep in mind the complex nature of these experiments, as well as nonspecific effects that may masquerade as antisense effects.

Is nectar reabsorption restricted by the stalk cells of floral and extrafloral nectary trichomes?

Reabsorption is a phase of nectar dynamics that occurs concurrently with secretion; it has been described in floral nectaries that exude nectar through stomata or unicellular trichomes, but has not yet been recorded in extrafloral glands. Apparently, nectar reabsorption does not occur in multicellular secretory trichomes (MST) due to the presence of lipophilic impregnations - which resemble Casparian strips - in the anticlinal walls of the stalk cells. It has been assumed that these impregnations restrict solute movement within MST to occur unidirectionally and exclusively by the symplast, thereby preventing nectar reflux toward the underlying nectary tissues. We hypothesised that reabsorption is absent in nectaries possessing MST. The fluorochrome lucifer yellow (LYCH) was applied to standing nectar of two floral and extrafloral glands of distantly related species, and then emission spectra from nectary sections were systematically analysed using confocal microscopy. Passive uptake of LYCH via the stalk cells to the nectary tissues occurred in all MST examined. Moreover, we present evidence of nectar reabsorption in extrafloral nectaries, demonstrating that LYCH passed the stalk cells of MST, although it did not reach the deepest nectary tissues. Identical (control) experiments performed with neutral red (NR) demonstrated no uptake of this stain by actively secreting MST, whereas diffusion of NR did occur in plasmolysed MST of floral nectaries at the post-secretory phase, indicating that nectar reabsorption by MST is governed by stalk cell physiology. Interestingly, non-secretory trichomes failed to reabsorb nectar. The role of various nectary components is discussed in relation to the control of nectar reabsorption by secretory trichomes.

Characterization of HDJ-2, a human 40 kD heat shock protein.

Heat shock proteins (HSP) are a large and complex family of proteins that play important roles in cellular function and survival. In previous studies, cDNA for a 45 kD human HSP (HDJ-2) was cloned and shown to be homologous to DNA-J, a bacterial HSP [F.M. Ausubel, R. Brent, R. E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1997; A. Chellaiah, A. Davis, T. Mohanakumar, Cloning of a unique human homologue of the Escherichia coli DNAJ heat shock protein, Biochim. Biophys. Acta 1174 (1993) 111-113]. We have also shown that the expression of HDJ-2 is highly elevated in kidney allograft biopsies of kidneys undergoing rejection [Y.G. Alevy, D. Brennan, S. Durriya, T. Howard, T. Mohanakumar, Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidneys, Transplantation 61 (1996) 963-967]. Because of the potential importance of HDJ-2 to disease pathogenesis, we carried out studies to characterize the structure and regulation of HDJ-2. Polyclonal and monoclonal antibodies that recognize recombinant HDJ-2 were prepared and used to localize its cellular expression. HDJ-2 was found to be farnesylated but not glycosylated. This HSP was ubiquitously expressed in all of the cell types we analyzed and was localized throughout the cytoplasm and around the nuclear membrane. However, upon heat shock it migrated to the Golgi, nucleolus, and the nuclear membrane. Northern blot analysis revealed two mRNA transcripts whose synthesis was not affected by heat shock. In addition, Western blot analysis showed that expression of HDJ-2 was also not affected by heat shock. Thus, our study shows the characterization of a HSP which, because of its migration pattern upon heat shock, is an excellent candidate for a protein chaperon.

Parathyroid hormone regulates the expression of the receptor protein tyrosine phosphatase, OST-PTP, in rat osteoblast-like cells.

Bone remodeling requires regulated tyrosine phosphorylation mediated by specific protein tyrosine kinases, such as c-src and c-fms, and to date, unknown protein tyrosine phosphatases (PTPs). We previously reported the isolation of a novel bone-specific receptor PTP, named osteotesticular PTP (OST-PTP), which is regulated during osteoblast differentiation and after exposure to PTH. To determine the relevance of this PTH regulation, we characterized the PTH-induced increase in OST-PTP messenger RNA (mRNA) in UMR 106 cells in comparison with PTH effects on a related receptor PTP and a PTH regulated gene, rat collagenase. Treatment of cells with rat PTH 1-34 (rPTH) resulted in a dramatic concentration and time-dependent increase in OST-PTP mRNA with a threshold at 4 h (= or < 1nM rPTH) and maximal response of 6- 10-fold above control levels at 8 h (100 nM rPTH). An increase in collagenase mRNA was detectable 2 h earlier at 100 pM rPTH with a maximal response at least 5-fold greater than that observed for OST- PTP. Levels of mRNA for the structurally similar PTP, rat leucocyte antigen-related molecule, were unaffected by rPTH treatment. Administration of cycloheximide (5-100 microM) abolished the OST-PTP and collagenase responses to PTH. The cAMP analogs, CPT-cAMP (0.01-1mM; 8 h) or Sp-cAMP (0.1 and 0.5 mM) were equal or greater in their effectiveness to enhance both OST-PTP and collagenase mRNA as compared with rPTH. In contrast, phorbol esters, calcium ionophore, bovine PTH (3-34), or human PTHrP (7-34) had no effect on either transcript. Interestingly, 36 h of pretreatment of cells with epidermal growth factor (10 ng/ml), a growth factor known to modulate PTH's actions, resulted in a significant decrease in the abundance of OST-PTP mRNA after rPTH exposure. These studies suggest that regulation of OST-PTP mRNA is a secondary response to PTH stimulation that is dependent on protein synthesis and that may be primarily by activation of the protein kinase A pathway. This specific modulation of a bone receptor PTP may prove to be a critical component in the PTH modulation of osteoblast function.

Construction and characterization of a bacterial clone containing the hemagglutinin gene of the WSN strain (HON1) of influenza virus.

A synthetic dodecadeoxynucleotide primer has been used to prepare a double-stranded DNA form of the hemagglutinin (HA) gene of a human influenza virus (WSN strain, HON1). This DNA has been inserted in plasmid pBR322 and cloned in bacterial cells. The insert contains nearly the complete hemagglutinin gene. A restriction map of this insert has been determined and structurally important areas of the HA gene have been sequenced. Amino acid sequences of several regions of the HA protein were deduced from the DNA sequences and compared to the known amino acid sequences of other influenza A viruses. WSN HA shows extensive homology to all influenza A viruses in a few regions, namely the first 17 amino acids of the N-terminus of HA1 (N-terminal polypeptide of HA) and the first 24 amino acids of the N-terminus of HA2 (C-terminal polypeptide of HA). The sequence diverges extensively from other influenza A viruses in most other areas. The sequence of WSN virus HA is similar to that of other HON1 viruses with the exception of the C-terminus of the HA1 peptide. The change in this area may contribute to some of the unique properties of WSN virus among the HON1 viruses. In addition, WSN HA contains a 17-amino-acid precursor before the N-terminus of HA1 and a single amino acid, arginine, connecting HA1 and HA2.

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli.

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.

Inhibition of GTP binding to Rac2 by peroxynitrite: potential role for tyrosine modification.

Peroxynitrite is a potent oxidant generated by the reaction of nitric oxide (*NO) and superoxide anion (O2*-), and both can be produced in inflammatory tissues. In the present studies, we analyzed the effects of peroxynitrite treatment on the GTP-binding activity of Rac2, a low molecular weight GTP-binding protein important in regulating a number of cellular functions. Using a fluorescent analog of GTP (methylanthraniloyl guanosine triphosphate or mant-GTP) as a reporter group, we found that treatment of Rac2 with peroxynitrite inhibited the binding of mant-GTP to Rac2 in a dose-dependent manner. Peroxynitrite was also able to react directly with free mant-GTP, resulting in a significant decrease in mant-GTP fluorescence; however, the mechanism of peroxynitrite-mediated damage to mant-GTP was different than with Rac2. In the case of mant-GTP, protection from peroxynitrite-mediated oxidation was observed in the presence of the free radical scavengers, mannitol and DMTU. In contrast, DMTU was unable to prevent peroxynitrite-mediated inhibition of mant-GTP binding to Rac2. Instead, our data demonstrates a role for peroxynitrite-mediated tyrosine modification in the inhibition of mant-GTP binding to Rac2, and we were able to demonstrate the formation of a significant level of nitrotyrosine formation in Rac2 exposed to peroxynitrite. Thus, our studies support the premise that oxidative modification of key cellular proteins, such as Rac2, plays an important role in the cytotoxic effects observed for peroxynitrite and other reactive oxidants.

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes.

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.

Toxicity, pharmacology and feasibility of administration of PEG-L-asparaginase as consolidation therapy in patients undergoing bone marrow transplantation for acute lymphoblastic leukemia.

We attempted to administer PEG-L-asparaginase (PEG-L-A) following hematologic recovery to 38 patients undergoing autologous or allogeneic marrow transplantation for acute lymphoblastic leukemia (ALL). Twenty-four patients (12 of 22 receiving allogeneic and 12 of 16 receiving autologous transplants) received between one and 12 doses of PEG-L-A, including nine who completed the planned 12 doses of therapy. The toxicities encountered were similar to those observed in non-transplanted patients undergoing therapy with PEG-L-A and included allergic reactions, pancreatitis, weight loss, hypoalbuminemia, and low levels of anti-thrombin III. Of the 24 who received the drug, eight remain in remission. Of 12 patients in second remission at the time of transplantation who received PEG-L-A, five of seven who received allogeneic and two of five who received autologous transplants remain in remission, 16+ to 46+ months from transplant. While PEG-L-A could be administered to most of the patients undergoing marrow transplantation for ALL, most patients either relapsed while receiving the drug or developed toxicities which resulted in abbreviated courses. At this time, we cannot recommend PEG-L-A as single agent, post-BMT chemotherapy.

Methotrexate compared with mercaptopurine for early induced abortion.

OBJECTIVE: To compare the antimetabolites methotrexate and 6-mercaptopurine as single-agent medical abortifacients using clinical and immunohistochemical analyses. METHODS: Twenty-seven women with gestations less than 7 weeks from the last menstrual period (LMP) were randomized to receive intramuscular methotrexate, 50 mg/m2, or oral 6-mercaptopurine, 200 mg. Forty-six additional women received methotrexate after randomization was discontinued. Women returned at 2-week intervals. Those without fetal cardiac activity were followed until complete abortion. Those with fetal cardiac activity were considered failures and underwent suction abortions. Tissue collected at the time of suction abortion was analyzed with the cell-proliferation immunohistochemical assay Ki-67. RESULTS: All 12 women in the 6-mercaptopurine group had fetal cardiac activity at follow-up and underwent suction abortion; therefore, this arm of the study was discontinued. Six of the 61 women who received methotrexate had fetal cardiac activity at follow-up and also underwent suction abortion. Fetal cardiac activity was present after methotrexate in three of 55 women at less than 6 weeks from the LMP and in three of six between 6 and 7 weeks from the LMP (P < .01). Women who aborted after methotrexate started bleeding on day 19 (standard deviation [SD] 7.8), bled for 9 days (SD 4.0), and used minimal pain medications. Tissues exposed to methotrexate showed decreased Ki-67 activity compared with tissues exposed to 6-mercaptopurine (P = .003). CONCLUSION: In oral doses of 200 mg, 6-mercaptopurine did not induce early abortion. A single intramuscular dose of methotrexate used without prostaglandins induced abortion in most women at gestational ages of less than 6 weeks. Ki-67 activity was lower in a small sample of fetal tissues exposed to methotrexate than in tissues exposed to 6-mercaptopurine.

Construction of adenoviral vectors.

Recombinant adenovirus vectors have proven to be useful tools in facilitating gene transfer. Construction of such vectors requires a knowledge of the adenovirus genome structure and its life cycle. A commonly used recombinant adenovirus involves deletion of the E1 region; such a recombinant is traditionally produced by overlap recombination after cotransfection of 293 cells with a plasmid shuttle vector and a large right-end restriction fragment of viral DNA. The shuttle vector contains a cassette for a transgene placed in region E1 and flanking sequences from adenovirus for recombination. Normally, a high background of parental virus results because of the difficulty in separating right-end restriction fragment length DNA from uncut DNA. This paper describes a negative selection based on the traditional cotransfection method using viral DNA from an E1-deleted adenoviral recombinant that expresses green fluorescent protein (GFP). In situ fluorescent microscopy is used to distinguish the recombinant plaques (white or nonfluorescent) from the parental virus plaques (green or fluorescent). In addition, this system allows for the detection of contaminating parental virus at later stages when production lots of the recombinant vector are being made.

The use of acute and preventative medical services by a general population: relationship to alcohol consumption.

AIMS: To investigate the hypothesis that increasing alcohol consumption is accompanied by increasing use of acute, but decreasing use of preventative, medical services among the general population. DESIGN AND PARTICIPANTS: Health and life-style survey of 41,000 randomly-sampled adults in SE England who self-completed a validated questionnaire covering socio-demographics, alcohol and tobacco usage and use of acute (A&E department and general practitioner) and preventative (dental, optician, mammography and cervical cytology) services: the response rate was 60%. MEASUREMENTS: Comparative use of acute and preventative health care services by patients with varying consumption of alcoholic beverages. This was estimated by the odds ratio for service use, after correcting for the following confounding variables; age, social class, ethnic group, employment status, whether lives with children or with other adults, whether is a career, limiting long-term illness, depression status, smoking habit and use of private health insurance. FINDINGS: There was increased use of accident and emergency services by the harmful and intermediate drinking groups compared with the safe drinking group. Male abstainers attended their A&E departments more frequently than 'safe limit' drinkers. With respect to preventative services, both male and female abstainers and harmful drinkers used dental services less than safe limit drinkers. For females, mammography and cervical cytology services were less frequently used by abstainers and by harmful drinkers. CONCLUSIONS: This study supports the generally held view that heavy alcohol consumers are disproportionate users of acute medical services but they are relative under-users of preventative medical care services. Alcohol abstainers are also over-users of acute services, but under-users of preventative services. These latter observations are relevant to the claims that moderate alcohol consumers have lower apparent morbidity and mortality rates compared to abstainers.

Topical steroid use in the treatment of ocular alkali burns.

BACKGROUND: Ocular alkali burns can be associated with a poor visual outcome. The release of collagenases and proteases after the injury leads to corneoscleral melting. The role of topical steroids in such patients is controversial as they have been postulated to exacerbate corneoscleral melting. METHODS: 30 patients were reviewed retrospectively after admission to King's College Hospital with alkali burns between 1990 and 1993. All patients were treated with an intense and prolonged regimen of topical steroids and topical and systemic vitamin C. RESULTS: 22 patients had mild injuries and eight had severe injuries as estimated by the Roper-Hall grading system. 23 patients were treated with topical steroids for > 10 days and 22 patients were treated with topical vitamin C for more than 10 days. One patient with a severe injury developed corneoscleral melting. CONCLUSION: Prolonged treatment with topical steroids when used in conjunction with topical vitamin C is not associated with corneoscleral melting.