Cloning and characterization of yeast Leu4, one of two genes responsible for alpha-isopropylmalate synthesis.

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.

Increased epidermal deoxyribonuclease I activity after clipping or plucking of hair and during wound healing.

An increase in extracellular epidermal DNase I occurs after slight to severe trauma to skin and appears to be generally associated with an inflammatory response including epidermal cell proliferation, rather than cell death. It precedes or accompanies the epidermal hyperplasia which occurs during healing of superficial wounds and regrowth of hair. It is suggested that extracellular epidermal DNase I is primarily of humoral origin and its increase after skin trauma results from increased vascular permeability and diffusion of a DNase I-inhibitor complex into the epidermis where the inhibitor is ultimately inactivated or destroyed.

Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage.

Using a combination of restriction endonuclease digestion, nuclease BAL 31 treatment, and standard ligation procedures, a 4.4-kb DNA segment that carried the yeast LEU4 gene [encoding alpha-isopropylmalate synthase (IPMS) I] and adjoining sequences was excised from an appropriate plasmid and replaced with the yeast HIS3 gene. The new plasmid was digested to obtain a linear HIS3-carrying fragment flanked by remnants of the LEU4 region. Integrative transformation of a LEU4fbr LEU5+ his3- strain with this fragment resulted in the deletion of the LEU4 gene from the genome of some recipients, as demonstrated by transformant phenotype, genetic analysis and the absence of RNA capable of hybridizing to a LEU4 probe. The leu4 deletion strains remained Leu+. The extract of one such strain contained about 18% of the IPMS activity of wild-type cells. It is concluded that the residual activity is that of a second IPMS (IPMS II) that depends on an intact LEU5 locus. IPMS II was inhibited by leucine, but its sensitivity was about an order of magnitude lower than that of IPMS I. Deletion of the LEU4 region by the method utilized here resulted in an amino acid auxotrophy that could be satisfied by methionine, homocysteine, or cysteine. Complementation tests and genetic analysis demonstrated that the affected gene was MET4. Linkage to MET4 would place the LEU4 gene on the left arm of chromosome XIV.

High dose rate afterloading intraluminal brachytherapy in malignant airway obstruction of lung cancer.

PURPOSE: This is a retrospective study to review the palliation rate, survival rate and complications of high dose rate (HDR) intraluminal brachytherapy in the treatment of malignant airway obstruction of lung cancer. METHODS AND MATERIALS: A total of 225 high dose rate (HDR) brachytherapy treatments were delivered to 76 patients with symptomatic malignant airway obstruction by remote afterloading technique. An average of 7 Gy at a radius of 1 cm from the center of the source was delivered by Iridium-192 (Ir-192) sources. The majority of the patients received 3 fractions at 2 week intervals. Fifty-four patients received HDR brachytherapy as part of their initial treatment; 20 patients presented as symptomatic endobronchial recurrence. Two patients received YAG laser photoresection to open up the obstruction to allow insertion of the brachytherapy catheter. Fifty-nine patients received concurrent external beam irradiation. Forty-two patients were given 60-70 Gy in 6-7 weeks with curative intent. Seventeen patients were given 20-59 Gy in 2-5 weeks as a palliative measure. Nine patients received a radiosensitizer. One patient received concurrent chemotherapy. RESULTS: The symptomatic response rates are as follows: dyspnea had an 87% response rate (59% partial response, 28% complete response), cough had a 79% response rate (47% partial response, 32% complete response), hemoptysis had a 95% response rate (38% partial response, 57% complete response), and postobstructive pneumonia had an 88% response rate (53% partial response, 35% complete response). Sixty-six patients had follow up endoscopic examination (1-3 months after brachytherapy). Their total response rate was 87% (52% partial response and 35% complete response). There were four acute complications: three cases of massive hemoptysis and one of mild hemoptysis. There are five late complications: three cases of radiation pneumonitis and two of esophagitis. At the time of this study, 55 patients have died with the maximum survival duration 113 months (9.4 years) from diagnosis date and 18 months from first HDR treatment. Twenty-one patients are still alive with a mean follow-up duration of 20 months from diagnosis date and 7.8 months from the first HDR treatment. CONCLUSION: HDR brachytherapy is an excellent modality for palliating symptomatic malignant airway obstruction with an acceptable complication rate; however, no definitive increase of survival rate was observed. Prospective clinical trials are needed to better define its merit regarding survival. This paper also includes a literature review and discussion of HDR brachytherapy on bronchogenic cancer.

The beta 1 sodium channel subunit modifies the interactions of neurotoxins and local anesthetics with the rat brain IIA alpha sodium channel in isolated membranes but not in intact cells.

Mammalian brain sodium channels consist of an alpha subunit and two smaller beta subunits. The role of the beta 1 subunit in modulating ligand interactions at these channels was examined using a cell line stably expressing human beta1 and rat brain IIA alpha subunits. Coexpression of the beta 1 subunit had no effect on the potencies of sodium channel blockers in inhibiting whole cell [3H]batrachotoxinin A benzoate ([3H]BTX) binding or veratridine-stimulated [14C]guanidinium influx. Coexpression of the beta 1 subunit also had no effect on the potencies of alpha scorpion toxin, brevetoxin, or RU 39568 in stimulating [14C]guanidinium influx. By contrast, coexpression of the beta 1 subunit had dramatic effects on ligand interactions in isolated membranes. In isolated membranes of cells expressing only the alpha subunit, the neurotoxins had no stimulatory effect on [3H]BTX binding and the potencies of local anesthetic-like channel inhibitors were 10-100-fold lower than those at native sodium channels. Whereas in membranes of cells coexpressing the beta 1 subunit, the neurotoxins increased [3H]BTX binding 30-fold and the potencies of the sodium channel inhibitors closely matched those found at native sodium channels. These findings indicate that the beta 1 subunit is not required for the binding of sodium channel activators or inhibitors but rather, that the beta 1 subunit may stabilize the alpha subunit in a functional conformation thereby allowing detection of these interactions in disrupted membranes.

Release of lysosomal hydrolases during simulated extracorporeal circulation.

Contact with surfaces results in activation of formed blood elements. This study demonstrates that during extracorporeal circulation, release of lysosomal enzymes occurs concomitantly with platelet granule secretion. Fresh, heparinized human blood was recirculated for 2 hours at 1,000 ml/min at 37 degrees C in silicone rubber circuits containing a membrane oxygenator (0.85 m2). The plasma levels of a platelet-specific protein, low affinity platelet factor 4 (LA-PF4), rose from less than 0.5 to 16 +/- 3 SEM microgram/ml plasma, indicating extensive release of platelet alpha granule contents. Concurrently, plasma activity of acid phosphatase and N-acetyl-beta-glucosaminidase increased nearly fivefold. Platelet inhibition prevented release of LA-PF4 and reduced acid phosphatase levels, but failed to alter the levels of N-acetyl-beta-glucosaminidase. Lidocaine (10 to 20 microgram/ml), however, prevented the rise in N-acetyl-beta-glucosaminidase activity and diminished acid phosphatase levels without altering secretion of LA-PF4. The failure of platelet inhibitors to block the increase in N-acetyl-beta-glucosaminidase, and the efficacy of lidocaine to do so without altering LA-PF4 secretion, suggest that leukocytes, not platelets, are the primary source of this lysosomal enzyme. Although acid phosphatase activity measured in plasma my be derived from leukocytes, platelet membranes, and not lysosomes, appear to be a more likely source. Release of hydrolytic enzymes from formed blood elements during cardiopulmonary bypass may cause endothelial cell injury and thus contribute to the increased vascular permeability associated with extracorporeal circulation.

Time course of thymoxamine reversal of phenylephrine-induced mydriasis.

We conducted a randomized, double-masked, paired comparison of 0.1% thymoxamine vs placebo for the reversal of phenylephrine-induced mydriasis. Mydriasis was induced with 2.5% phenylephrine in each eye of 74 subjects (148 eyes). Each subject then received 0.1% thymoxamine in one eye and placebo in the other eye. Pupillary measurements were obtained at regular intervals during the ensuing 8 hours. At all intervals, a greater percentage of thymoxamine-treated eyes returned to baseline pupillary diameters compared with placebo-treated eyes (P less than or equal to .01). For subjects in whom both pupils returned to baseline, thymoxamine-treated eyes returned to baseline in a mean of 2.2 hours, vs 5.2 hours for placebo (P less than .0001). Among thymoxamine-treated eyes, those with light irides responded more rapidly than those with dark irides, returning to baseline in 1.6 vs 2.8 hours, respectively (P = .0046). After constriction to baseline pupillary diameter had been achieved, no patients experienced a rebound dilation.

New imidazoles as probes of the active site topology and potent inhibitors of beta-glucosidase.

Series of 4-arylimidazoles, omega-N-acylhistamines and 4-(omega-phenylalkyl)imidazoles were synthesized in order to probe the active site topology of sweet almond beta-glucosidase. These imidazole derivatives were shown to be very powerful competitive inhibitors. Among the 20 tested compounds, omega-N-benzoylhistamine and 4-(3'-phenylpropyl)imidazole are the most potent inhibitors of the enzyme, with pH-independent Ki values of 0.06 and 0.07 microM, respectively. The inhibition of 4-(omega-phenylalkyl)imidazoles exhibited an interesting trend as to Ki values: 4-phenylimidazole (6.6 microM)>4-benzylimidazole (1.4 microM)>4-(2'-phenylethyl)imidazole (0.82 microM)>4-(3'-phenylpropyl)imidazole (0.07 microM)<4-(4'-phenylbutyl)imidazole (0.13 microM)<4-(5'-phenylpentyl)imidazole (0.3 microM). This revealed that the imidazole and aryl binding sites (which result from favorable interactions within the corresponding glycone and aglycone binding subsites) are separated by the optimal distance equivalent to the length of a -CH2-CH2-CH2- group. Substitutions of the phenyl moieties of 4-phenylimidazole and 4-benzoylhistamine result in weaker inhibition. These classes of imidazoles are particularly powerful inhibitors of sweet almond beta-glucosidase.

Comparison of radiation therapy of classic and epidemic Kaposi's sarcoma.

Between 1963 and 1990, 92 lesions in twenty patients with Kaposi's sarcoma (KS) were treated using radiation therapy (RT). Fifty-nine classic Kaposi's sarcoma (CKS) lesions in 12 patients were treated with 990 cGy in 3 fractions-5,000 cGy in 25 fractions; 39 lesions (66%) showed a complete response (CR), 15 (25%) showed a partial response (PR) with complete symptomatic relief, and 5 lesions (8%) showed no response (NR). Only 6 of 59 lesions (10%) recurred in field with a median recurrence-free duration of 8 months. Thirty-three epidemic Kaposi's sarcoma (EKS) lesions in 8 patients were treated from 1,000 cGy in 5 fractions-3,000 cGy in 10 fractions. Thirty-one EKS lesions (94%) showed CR, and two lesions (6%) showed PR. An extensive review of the literature is also presented in the study here reported.

Prenatal diagnosis of trisomy 20 mosaicism by maternal serum screening for Down syndrome.

We report two cases of prenatally diagnosed trisomy 20 mosaicism associated with positive Down syndrome screening at 16 weeks' gestation. Both infants exhibited normal growth and mental development. These cases suggest that the multiple-marker screening test may play an important role in prenatal detection and diagnosis of chromosomal anomalies in addition to Down syndrome.

Optimized combinations of abatement strategies for urban mobile sources.

The maximum incremental reactivity (MIR) scale was chosen as a practical index for quantifying ozone-forming impacts. The integer linear and nonlinear programming techniques were employed as the optimization method to maximize MIR and volatile organic compound (VOC) reductions, and minimize ozone's marginal cost with varied control costs. Mobile vehicles were divided into nine categories according to the demands of decision makers and the distinctive features of local circumstance in metro-Taipei. The emission factor (EF) and vehicle kilometers traveled (VKT) of each kind of vehicle were estimated by MOBILE5B model via native parameters and questionnaires. Compressed natural gas (CNG) and inspection and maintenance (I/M) were the alternative control programs for buses and touring buses; liquefied petroleum gas (LPG), I/M, methanol, electrical vehicle (EV) were for taxis and low duty gasoline vehicles. EV, methanol, and I/M were the possible control methods for two-stroke and four-stroke engine motorcycles; I/M programs for low-duty diesel trucks, heavy-duty diesel trucks, and low-duty gasoline trucks. The results include the emission ratios of specific vehicle to all vehicles, the best combination of abated measures based on different objectives, and the marginal cost for ozone and VOC with varied control costs.

[Effect of naomaitong on basilar artery rings and portal vein strips].

Both Naomaitong and single salvia miltiorrhiza inhibit the contraction of rabbit basilar artery rings evoked by CaCl2 and KC1 and have nonspecific antagonism against the quantity effect curve of CaCl2. Naomaitong and single salvia miltiorrhiza also inhibit the contraction of rabbit basilar artery rings evoked by KC1. Clearly the inhibitory function of the former is greater than that of the latter. But on the myogenic activity of portal vein strips in rats Naomaitong has light inhibitive effect while salvia miltiorrhiza has not.

Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogues.

The structure of the GTP-binding site of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and include 8-azido-GTP, P3-(4-azidoanilino)-P1-5'-GTP (AA-GTP), 5'-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)-GTP (ANPAP-GTP), the 2',3'-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5'-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2'- and 3'-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T alpha subunit of transducin and was localized on the 21-kDa tryptic fragment of T alpha. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T alpha and T beta gamma subunits of transducin. The labeling on T alpha was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T alpha and T beta gamma. This observation implies that the guanine ring of the bound GTP on T alpha could be in close proximity with T beta gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of common variant alleles of the human guanosine monophosphate reductase gene.

Examination of nucleotide sequences of genomic DNA samples obtained from several unrelated Caucasians and orientals revealed the existence of four variant alleles in the chromosome 6-linked quanosine monophosphate reductase locus. The wild-type gene has T at position 42 (counting from A of the chain initiation codon), C at 630, G at 700, and T at 766, i.e., its structure is T(42)-C(630)-G(700)-T(766). The variant gene, T-T-G-T, was found in about 10% of the loci examined. The C-to-T change at 630 was silent and did not induce any amino acid substitution (His at amino acid residue 210), but it created an additional NcoI cleavage site in the variant gene. The frequency of another variant, the T-C-G-A gene, was about 30%. The T-to-A change at 766 caused an amino acid substitution Phe----Ile at amino acid residue 256 in the variant protein. Frequencies of the C-C-G-T variant and the T-C-A-T variant were probably lower than 5% in Caucasians and orientals.

Central retinal vein occlusion associated with retinal arteriovenous malformation.

PURPOSE/BACKGROUND: Retinal arteriovenous malformations can be seen in a variety of ways and have multiple, associated, ocular changes that can affect vision. The authors report two cases of retinal arteriovenous malformation. In each case, a central retinal vein occlusion developed. METHODS: Each patient underwent clinical examination and fluorescein angiography. One patient was followed over a long period of time. CONCLUSION: The authors propose that a turbulent flow, high intravascular volume, and arteriolar pressure in the venous side of the retinal arteriovenous malformation may lead to vessel wall damage, thrombosis, and occlusion. They also suggest that compression of the central retinal vein by the mass effect of the arteriovenous malformation on the optic nerve further leads to turbulence and thrombosis.

Terbutaline nebulization and epinephrine injection in treating acute asthmatic children.

Ninety children with acute asthma, equally divided into two study groups, were studied to compare the efficacy and safety of nebulized terbutaline with injected epinephrine in the treatment of acute exacerbation. The terbutaline group received 2 ml (5,0 mg) terbutaline solution diluted with 2 ml 0.9% saline for inhalation over 10 minutes; the epinephrine group received 0.01 ml/kg of 1:1000 epinephrine (maximum 0,3 ml) through subcutaneous injection at deltoid area. Spirometry, pulse oximetry, and clinical severity scoring system were evaluated at baseline and again 15 minutes after treatment. The baseline data of the two groups were not significantly different. The clinical severity score and spirometry of both groups were significantly improved after treatment. Compared with the terbutaline group, the epinephrine group had better mean oxygen saturation (SaO2; p < 0.001), frequency of oxygen desaturation (p = 0.0028) and forced expiratory flow 25-75% (FEF25-75%, p = 0.027). For those patients with initial forced expiratory volume in one second (FEV1) lower than 60% of predicted value, epinephrine treatment was more effective in the improvement of FEV1, FEF25-75%, and oxygen saturation (SaO2) (p = 0.011, 0.012, and 0.006, respectively). A Significantly higher rate of adverse effects occurred in patients given epinephrine (47% vs 11%, p = 0.0002); these included pallor, tremor, dizziness, headache, palpitation, soreness of legs, numbness of extremities, cold sweating, general weakness and nausea. Considering the general trend to noninvasive therapy in children and the more frequent adverse effects after epinephrine injection, such nebulized beta-2 agonists as terbutaline appear preferable for initial therapy of acute asthma if oxygen is supplemented to prevent possible hypoxemia. However, parenteral epinephrine still is worth trying, particularly in any severe, life-threatening attack.