Gene discovery and gene function assignment in filamentous fungi.

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Vitamin E ameliorates adverse effects of endothelial injury in brain arterioles.

Endothelium-dependent dilation, produced by applying acetylcholine (ACh) to pial arterioles, was unaffected after 6 mo of a diet with zero vitamin E or 8 mo of a vitamin E-enriched diet. The enriched diet did not affect constriction produced by topically applied NG-monomethyl-L-arginine, an inhibitor of the synthesis of endothelium-derived relaxing factor (EDRF). EDRF mediates the response to ACh and is a basally released dilator and antiplatelet paracrine substance. Endothelial injury produced by a helium-neon laser and Evans blue technique eliminates the response to ACh, but in vitamin E-enriched mice the response to ACh was unaffected by the injury. More prolonged exposure of the laser induces platelet adhesion/aggregation at the injured site. A significantly longer exposure to the laser was required to initiate adhesion/aggregation in vitamin E-enriched mice. Because effects of endothelial damage in this model are mediated at least in part by singlet oxygen produced by injured tissue (W.I. Rosenblum and G.H. Nelson, Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1258-H1263, 1996.), we conclude that the antioxidant, radical-scavenging actions of vitamin E explain the protective action of the vitamin E-enriched diet. However, raising vitamin E levels did not protect against putative adverse effects of normally occurring oxidants.

Murine red blood cell fragility is not affected by either vitamin E depletion or supplementation.

Male ICR mice were Pair-fed semipurified diets containing 0, 55 (control), and 500 IU/kg of vitamin E. Plasma and hepatic concentrations of vitamin E were determined and found to parallel the vitamin E levels in the diet. Even though plasma vitamin E levels were virtually zero in mice fed the depleted vitamin E diet for up to 304 days, there was no statistical difference in the red blood cell fragility between these animals and controls, as determined by a hypoosmotic fragility test. The diet with enriched vitamin E concentrations also did not affect the fragility of the red blood cell (RBC). Even after 300 days of zero dietary vitamin E, mice appeared healthy, demonstrating neither neurologic dysfunction nor failure to thrive. The data indicates that mice, unlike several other species, are more resistant to vitamin E depletion and may have other mechanisms to compensate for loss of this important antioxidant.

Role of platelet-endothelial cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo and ex vivo.

BACKGROUND AND PURPOSE: We previously demonstrated that a monoclonal antibody (MoAb) with anti-CD31, anti-platelet-endothelial cell adhesion molecule (PECAM)-like properties delayed platelet adhesion/aggregation at a site of minor endothelial injury. To our knowledge, this was the first in vivo demonstration of an effect of anti-CD31. There was no exposure of collagen or basal lamina at the injured site, and the modulation of adhesion/aggregation at such sites has not received much study. The present investigation attempted to replicate the first with the use of a different MoAb, definitely characterized as anti-PECAM. In addition, an ex vivo investigation was performed to see whether the in vivo action of anti-PECAM could have been caused by an effect of the MoAb on the platelets rather than on the endothelium. METHODS: A helium-neon laser, in the presence of intravascular Evans blue, was used to injure the endothelium of arterioles on the surface of the mouse brain. Intravital microscopy was used to determine the number of seconds required for the light to initiate the first recognizable platelet aggregate forming at the injured site. Mice injected with vehicle were compared with mice injected with 2 mg/kg anti-PECAM through the tail vein. The injection was 10 minutes before challenge with the laser. Additional studies were performed of aggregation produced in vitro by arachidonate and by ADP added to platelet-rich plasma (PRP) prepared from blood taken from MoAb-treated and vehicle-treated mice. RESULTS: Aggregation latency was significantly prolonged (P<.02) by anti-PECAM (121+/-59 versus 65+/-26 seconds in controls; n=10 each). Aggregation ex vivo was not affected. CONCLUSIONS: PECAM is an important modulator of platelet adhesion/aggregation at sites of minor endothelial damage in brain arterioles. The data are consistent with the hypothesis that PECAM sites on the endothelium are involved and may be exposed by the injury to promote adhesion/aggregation. Since the endothelial cell layer is intact at these sites, mechanisms such as this offer important alternatives to the more commonly studied pathways of platelet activation, which require exposure of collagen and are not applicable in this model.

Singlet oxygen scavengers affect laser-dye impairment of endothelium-dependent responses of brain arterioles.

This study investigates the possible role of singlet oxygen in accounting for the inhibitory effect of laser-dye injury on endothelium-dependent dilations. The combination of helium-neon (HeNe) laser (20-s exposure) and intravascular Evans blue impairs endothelium-dependent dilation of mouse pial arterioles by acetylcholine (ACh), bradykinin (BK), and calcium ionophore A23187. Each has a different endothelium-derived mediator (EDRFACh, EDRFBK, EDRFionophore, respectively). In this study, diameters at a craniotomy site were monitored in vivo with an image splitter-television microscope. The laser-dye injury, as usual, abolished the responses 10 and 30 min after injury, with recovery, complete or partial, at 60 min. Dilations by sodium nitroprusside, an endothelium-independent dilator, were not affected by laser-dye. When the singlet oxygen scavengers L-histidine (10(-3) M) and L-tryptophan (10(-2) M) were added to the suffusate over the site, the responses to ACh at 10 and 30 min were relatively intact, the response to BK was partly protected at 10 min only, and the response to ionophore was still totally impaired at 10 and 30 min. Lysine, a nonscavenging amino acid, had no protective effects with any dilator. We postulate that a heat-induced injury initiates a chain of events resulting in prolonged singlet oxygen generation by the endothelial cell (not by the dye). We postulate further that destruction of EDRFACh by singlet oxygen is responsible for laser-dye inhibition of ACh and that generation of the radical must continue for > or = 30 min. On the other hand, the heat injury itself is probably responsible for the elimination of the response to ionophore. Heat plus singlet oxygen generated by heat-damaged tissue may initially impair the response to BK, but by 30 min only the effects of some other factor, presumably heat injury, account for the impaired response to BK.

Intrauterine diagnosis of hydrothorax in a fetus who had a combination chylothorax and pulmonary sequestration after delivery.

Congenital hydrothorax was diagnosed prenatally by transabdominal ultrasonography. Five fetal thoracentesis procedures were performed and biochemical analyses of the fluids were done. After delivery a combination chylothorax and extralobar pulmonary sequestration was demonstrated in the infant. We believe this to be the first case report of this combination investigated prenatally. We suggest that investigators perform biochemical analyses on fetal pleural fluid removed in cases with a single diagnosis. Perhaps by comparing those data with the data reported here, a biochemical marker can be identified that will be useful in distinguishing these two conditions in utero.

Delayed platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles after transient elevations of blood pressure and shear.

BACKGROUND AND PURPOSE: Prior research showed that injection of angiotensin II (Ang II) produces a transient elevation of blood pressure (BP) and shear in pial arterioles. This inhibited platelet adhesion/aggregation at a site of subsequently injured endothelium. The present study attempted to confirm the Ang II finding with a different method of endothelial injury, to test the hypothesis that the effect on adhesion/aggregation was a consequence of prolonged release of "classic" endothelium-derived relaxing factor (released by acetylcholine [Ach]; EDRFACh) produced by the preceding transient elevation in shear, and to show with the use of norepinephrine rather than Ang II that the effect of a preceding elevation of BP was independent of the pressor agent used. METHODS: Focal platelet adhesion/aggregation was elicited in cerebral surface (pial) arterioles by producing minimal endothelial damage with a helium-neon laser/Evans blue dye technique. Vessels were observed by intravital microscopy. We recorded the time required for the laser to elicit adhesion/aggregation in control mice and in mice given Ang II in a dose of 16 micrograms/25 g IP. This dose produces an abrupt and significant elevation of BP and shear, which return to baseline levels in less than 30 minutes. Laser/dye damage of endothelium and resultant adhesion/aggregation of platelets were not induced until after BP and shear returned to basal levels. The effect of topical Ang II on damage-induced adhesion/aggregation was also tested. In addition, mice injected with Ang II were treated with either topical indomethacin 40 micrograms/mL or topical Ng-monomethyl L-arginine (L-NMMA; 10(-6) mol/L) in an effort to prevent the preceding increase in shear from inhibiting subsequent adhesion/aggregation. Finally, norepinephrine instead of Ang II was used to transiently raise BP (and shear) in an effort to delay subsequently induced adhesion/aggregation. RESULTS: Platelet adhesion/aggregation at the injured site was significantly delayed by a prior transient rise in shear produced by either Ang II or norepinephrine. Locally applied Ang II failed to influence adhesion/aggregation, although a previous study showed that such Ang II reaches the endothelium. Locally applied indomethacin had no effect on inhibition of platelet adhesion/aggregation, but locally applied L-NMMA prevented the prior transient elevation of shear from inhibiting adhesion/aggregation at a subsequently injured site. CONCLUSIONS: Elevation of BP with consequent elevation of shear inhibits local platelet adhesion/aggregation even when the latter is initiated by endothelial damage produced after return of shear to basal levels. The direct action of Ang II on endothelium is not responsible for the effect on adhesion/aggregation, and indeed the effect is independent of the pressor agent. The pharmacological data, together with the literature, support the hypothesis that increased shear causes an increased release of EDRFACh, which may continue for at least some minutes after return of shear to normal levels.

Protein synthesis and rapid recovery of endothelium-dependent dilation after endothelial injury of pial arterioles.

A laser-dye technique is known to injure selectively microvascular endothelium in situ. With the use of the arterioles on the surface of the mouse brain (pial arterioles), the endothelial injury is manifest by loss of several endothelium-dependent responses, including the dilation produced by topical application of acetylcholine (ACh), bradykinin (BK), and calcium ionophore A-23187. The responses normally recover by 60 min following injury. The present study shows that inhibitors of protein synthesis prevent the recovery. Either actinomycin D (Act-D; 1 mg/kg) or cycloheximide (CX; 10 mg/kg) was injected intraperitoneally 35 min before the light-dye injury. The CX was used in separate studies with ACh, BK, and ionophore. In each study, recovery of the endothelium-dependent dilation failed to occur 60 min after injury, while recovery occurred in contemporary vehicle-treated controls. Act-D was used in separate studies of ACh and BK. Again, recovery of endothelium-dependent dilation was prevented. Neither CX nor Act-D inhibited the response to ACh in uninjured vessels. Thus response to ACh was intact in such arterioles 90 min after the injection of CX or Act-D. CX was also given to uninjured mice and was found to have no inhibitory action on the response to ionophore 90 min later. CX and Act-D inhibit protein synthesis by very different mechanisms, inhibition of translation and of transcription, respectively. Because both prevented recovery of the endothelium-dependent responses, we conclude that one or more rapidly synthesized proteins are required for the recovery.

Anti-CD31 delays platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles.

The arterioles on the surface of the mouse brain (pial arterioles) were observed by in vivo microscopy. A focus of minor endothelial damage was produced in a single pial arteriole in each mouse by briefly exposing the site to a helium neon laser after an intravenous injection of Evans blue. Mice were injected 10 minutes before injury with a monoclonal antibody (MAb) to mouse CD31, also known as platelet endothelial cell adhesion molecule. This treatment doubled (P < .01) the time required for the laser to produce a recognizable platelet aggregate. In additional experiments, an MAb to mouse CD61 and an MAb to mouse intercellular adhesion molecule 1 had no effect. The data support previous observations indicating that platelet adhesion/aggregation in this model is induced by endothelial injury without exposure of basal lamina. The data are consistent with the hypothesis that the endothelial injury exposes or activates a platelet endothelial cell adhesion molecule on the endothelium which is blocked by the MAb directed against CD31. This may be the first demonstration of an effect of an anti-platelet endothelial cell adhesion molecule on platelet endothelial cell adhesion molecule on platelet adhesion/aggregation in vivo.

Comparison of transvaginal sonography with endometrial biopsy in asymptomatic postmenopausal women.

The purpose of this study was to compare TVS with endometrial biopsy as a screening technique in asymptomatic postmenopausal women. Asymptomatic postmenopausal women were recruited by newspaper advertisement. Each study patient was subjected to pelvic examination and TVS followed by endometrial biopsy. Patients with suspected endometrial abnormalities by TVS (normal by endometrial biopsy) were evaluated further with hysteroscopy with biopsy or D&C or both. Eight patients were identified as having abnormalities by TVS, only one of whom had abnormalities by initial endometrial biopsy. Two patients were identified as having abnormalities by endometrial biopsy and normal by TVS. Further evaluation of the seven patients identified as having abnormalities by TVS (normal by endometrial biopsy) documented all seven patients as having abnormalities. The total yield of abnormalities with TVS was 16% (eight of 50 patients). The total yield of abnormalities with endometrial biopsy was 6% (three of 50 patients). The sensitivity of TVS in identifying endometrial abnormalities was 80% (eight of 10), while endometrial biopsy was only 30% (three of 10). TVS was more sensitive in detecting endometrial abnormalities, including endometrial hyperplasia, than was endometrial biopsy. The use of endometrial biopsy as a screening technique in asymptomatic postmenopausal patients is questioned.

Correlation between the clinical diagnosis of bacterial vaginosis and the results of a proline aminopeptidase assay.

OBJECTIVE: The object of this study was to develop a simple and inexpensive test for detection of bacterial vaginosis (BV) in pregnant patients and to test its accuracy in a clinic population. METHODS: We developed a modified proline aminopeptidase (PAMP) assay to detect BV and compared the results of the assay with the clinical diagnosis of BV. RESULTS: The results of the PAMP assay in 55 asymptomatic and 50 symptomatic subjects significantly correlated with a clinical diagnosis of BV. The prevalence of BV in the asymptomatic population was 42% (PAMP assay) and 38% (clinical diagnosis). In the symptomatic population, it was 50% (PAMP assay) and 54% (clinical diagnosis). The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the PAMP assay were 86, 85, 86, 78, and 91%, respectively, in asymptomatic patients and 89, 96, 92, 96, and 88%, respectively, in symptomatic patients. CONCLUSIONS: The modified PAMP assay, which we describe, met our goals for simplicity, cost, and accuracy. We feel it could be best used as a screening test for BV in asymptomatic pregnant patients.

Correlations between morphological changes in platelet aggregates and underlying endothelial damage in cerebral microcirculation of mice.

BACKGROUND AND PURPOSE: The aim of this study was to test the hypothesis that, once formed, platelet aggregates may injure underlying cerebrovascular endothelium. Such injury could make the same site selectively attractive to the next wave of passing emboli or activated platelets. This vicious circle could account for repetitive, stereotypic symptoms in transient ischemic attacks. METHODS: In pial arterioles, minor endothelial injury was produced by a laser/dye technique. After various periods of platelet aggregation at the damaged site, the vessels were fixed in situ for electron microscopic study. The degree of platelet activation (rounded and/or degranulated forms) was evaluated by counting these forms in the electron photomicrographs. These counts were related to the degree of endothelial damage ascertained in the micrographs. Other statistical relations were also examined. RESULTS: Endothelial damage progressed in parallel with the duration of platelet aggregation and the degree of platelet degranulation at the site. Correlations were number of activated platelets versus degree of damage, r = .43, P < .03; duration of aggregation versus damage, r = .52, P < .01; and number of degranulated platelets versus the degree of endothelial damage, r = .83, P < .001. If an aggregate embolized, endothelial damage did not appear to progress. No correlation existed between the duration of exposure to the laser and the degree of injury. CONCLUSIONS: The parallel between changes in platelets and endothelial damage could represent either an effect of endothelium on platelets or an effect of platelets on endothelium. Although the former alternative cannot be totally ruled out, the observations seem to fit best the hypothesis that progressive endothelial damage can result from increasing activation and degranulation of overlying platelets.

Estimation of uterine volume with ultrasound in women with endometrial carcinoma.

In calculating uterine volume with ultrasound, it is necessary to calculate the volume of a parallelogram containing the uterus or to assume a specific shape for the uterus. The objective of this study was to determine how much (what percentage) of the parallelogram was actually occupied by the uterus without assuming any specific shape for the uterus. Overall, the volume of the uterus occupies 63% of the volume of the parallelogram. The uterus occupies a greater percentage of smaller parallelogram volumes than larger ones. The data also suggest that the concomitant presence of myomata may be responsible for the positive intercept of the regression line and therefore for the finding that small uteri occupy more than 63% of the parallelogram volume.

Endothelium-dependent effects of substance P and calcitonin gene-related peptide on mouse pial arterioles.

BACKGROUND AND PURPOSE: The effects of substance P (SP) and calcitonin gene-related peptide (CGRP) were tested on pial arterioles of mice. This was done because (1) perivascular peptidergic nerves may play an important role in modulation of cerebrovascular responses; (2) there are conflicting data concerning the mechanism of action of CGRP; (3) there are few or no studies directly testing the endothelium dependence of dilation by these peptides in the cerebral circulation; and (4) we wished to extend previous observations of mice by comparing peptidergic responses in the mouse with those published for other species. METHODS: The pial arterioles were monitored in vivo using video microscopy and image-shearing techniques for measuring diameter. Focal endothelial injury was produced with a laser-Evans blue technique. Responses to SP and CGRP were tested before and after endothelial injury. They were also tested before and during treatment with agents that interfere with responses mediated by endothelium-derived relaxing factor (EDRFACh). They were also tested before and during treatment with indomethacin. RESULTS: Both CGRP and SP produced dilation that was blocked by endothelial injury and by agents interfering with responses mediated by EDRFACh. Indomethacin had no effect. CONCLUSIONS: SP and CGRP produce endothelium-dependent dilations. These dilations are probably mediated by EDRFACh. With respect to SP, these results are similar to those reported for other vessels and species. With respect to CGRP, the finding of endothelium dependence has not been previously reported for cerebral vessels. However, very few species have been tested. Reports of other vascular beds in other species sometimes parallel and sometimes contradict our findings with CGRP.

Chorionic villus sampling in a patient who is an unusual carrier of hemophilia B.

We describe a patient who is a carrier of hemophilia B, who was unusual in that she had symptoms and abnormal hematologic findings. She became pregnant and desired to have chorionic villus sampling for fetal sex determination. This was performed without complication. Her pregnancy continued, and she was delivered of a normal female infant with no complications for mother or infant. We believe this to be the first report of chorionic villus sampling in a symptomatic carrier of hemophilia B.

M3 and M1 receptors in cerebral arterioles in vivo: evidence for downregulated or ineffective M1 when endothelium is intact.

Pial arterioles of mice were monitored by intravital television microscopy. Responses to acetylcholine (ACh) and to McN-A-343, a selective M1 activator, were monitored before and after endothelial injury by a laser-dye technique in the presence or absence of different reference antagonists to the M1, M2, and M3 muscarinic receptors. ACh and McN-A-343 produced endothelium-dependent dilation that was blocked by 4-diphenyl-N-methyl-piperidine methiodide, an inhibitor of the M3 receptor. Dilations were not augmented by blocking the M1 receptor with pirenzepine. Endothelial injury unmasked a constricting effect of both ACh and McN-A-343. The constricting effect was blocked by pirenzepine. The results support the literature suggesting an action of McN-A-343 at more than one site. They indicate that ACh causes endothelium-dependent relaxation via the M3 receptor and directly constricts vascular smooth muscle via the M1 receptor. There does not appear to be continuous competition between endothelium-dependent relaxation and endothelium-independent constriction. Rather the M1 receptors mediating constriction in vascular smooth muscle appear downregulated and/or uncoupled from the contractile machinery as long as the overlying endothelium synthesizes/releases the mediator (endothelium-derived relaxing factor) of ACh's dilating action.

Interaction of endothelium with dilation produced by inhibitors of cyclic nucleotide diesterases in mouse brain arterioles in vivo.

BACKGROUND AND PURPOSE: In vitro evidence gathered from extracerebral conductance vessels suggests interaction between the endothelium-derived relaxing factor for acetylcholine (EDRFACh) and cyclic nucleotide action in vascular smooth muscle. The purpose of this study was to examine this interaction in vivo in pial arterioles. As had been done in vitro, we used phosphodiesterase inhibitors that elevate cyclic nucleotide levels in vascular smooth muscle. METHODS: Pial vessels of mice were observed with television microscopy. Diameter of the arterioles was monitored with an image-splitting technique. The responses to topically applied phosphodiesterase inhibitors were tested before and after focal endothelial injury or before and during application of N-guanidino-L-monomethyl arginine (L-NMMA). Both treatments are known to eliminate the endothelium-dependent response to acetylcholine in this preparation. RESULTS: Phosphodiesterase inhibitors dilated pial arterioles. This was true for phosphodiesterase inhibitors elevating levels of both adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) as well as for inhibitors thought to selectively affect either nucleotide. The dilation was inhibited by endothelial injury and by L-NMMA. CONCLUSIONS: The data suggest that brain arterioles are dilated by both cAMP and cGMP but that this action is impaired if EDRFACh levels are reduced. Since EDRFACh elevates cGMP levels, these data support the hypothesis that the relaxing actions of cGMP and cAMP depend upon adequate basal levels of cGMP in vascular smooth muscle. This hypothesis, originally introduced in studies of extracerebral conductance arteries in vitro, can now be applied to brain resistance vessels in vivo.