Photoacoustic injury and bone healing following 193nm excimer laser ablation. The argon-fluoride excimer laser was investigated as a cutting-ablating tool for bone surgery. A total of 52 rats were divided into two experimental groups and two control groups. In one experimental group cortical bone defects were made; in another experimental group defects penetrating into the medullary space were performed. In the two control groups similar defects were achieved using water-cooled carbide burs. The rats were sacrificed on each of the 3, 7, 10, 20, 30, and 40 postoperative day. The cortical bone, the medullary space, and the extrabony tissue were examined by means of light microscopy. In both experimental groups, bone damage, represented by osteocyte destruction, extended to 1,050-1,450 microns ahead from the irradiated site, and bone healing was very much impaired. In the control groups no histological changes could be identified and bone healing appeared to be within normal limits. We believe this extensive bone damage, following 193 nm irradiation, to be a result of photoacoustic waves propagating in the bone following each pulse. In view of our results we feel that excimer lasers presently in use are not suitable for bone surgery. This problem of photoacoustic damage can be overcome in one of two ways: by designing a CW excimer laser or by reducing the pulse width to the picosecond regime.


Feasibility of skin deepithelialization using the contact neodymium-yttrium-aluminum-garnet laser. In a manner similar to laserbrasion, the carbon dioxide (CO2) laser has a proven role for expediting skin deepithelialization, which is an essential component of many reconstructive procedures. However, the contact Neodymium-Yttrium-Aluminum-Garnet (Nd:YAG) laser has many additional attributes such as tactile feedback and less plume that theoretically offer an advantage over the CO2 laser for this adjunctive maneuver. In rat experimental models and clinical applications, removal of the epidermis necessary for skin deepithelialization may indeed be achieved using a rounded probe for vaporization with the contact Nd:YAG laser. Unfortunately, the length of time necessary for skin deepithelialization with the Nd:YAG laser exceeded even conventional methods, such that this role with the present technology, although possible, is inefficient and impractical.


Effects of coaxial CO2/Nd:YAG irradiation on periodontal wound healing. This study evaluated a coaxial CO2/Nd:YAG laser used during periodontal surgery in mongrel dogs for the purpose of ablating the osseous portions of root surfaces to increase reattachment of soft tissue. Periodontal defects were produced with ligatures of orthodontic wire and elastics. The left mandibular osseous root surfaces were irradiated with a defocused, CW laser beam [power density (PD) = 42.46 W/cm2, energy density (ED) = 424.63 J/cm2]. The left maxillary osseous root surfaces were treated with a beam at twice the energy density [PD = 84.93 W/cm2, ED = 849.3 J/cm2]. The mandibular right quadrant received conventional surgery and the maxillary right quadrant served as the untreated control. Fourteen days after treatment, facial/lingual attached gingivae were scored for soft tissue adherence to bone. Laser irradiation at low ED did not improve soft tissue attachment when compared to conventional treatment. Higher ED irradiation produced significantly less attachment and increased tissue necrosis.


Microprocessor-controlled Nd:YAG laser for hyperthermia induction in the RIF-1 tumor. Near-infrared radiation from a Nd:YAG laser at 1,064 nm was used interstitially or superficially to induce hyperthermia in RIF-1 tumors in C3H male mice. A single 600-microns quartz fiber with a 0.5-cm cylindrical diffusor or a weakly diverging microlens at its distal end was used to deliver laser energy to tumors in the hind leg (mean volume = 100 mm3). Two thermocouples were inserted into each tumor. One thermocouple controlled a microprocessor-driven hyperthermia program (maximum output of 3.5 Watts) to maintain the desired temperature. Tumors were exposed to various temperature-time combinations (42-45 degrees C/30 min). Our initial results indicated that excellent temperature control to within 0.2 degrees C of the desired temperature at the feedback thermocouple was achievable during both superficial and interstitial heat treatments. Temperatures at the second thermocouple, however, were found to be lower by as much as 2.3 degrees C (using the cylindrical diffusor) or higher by up to 4.6 degrees C (using the microlens) when compared to the feedback thermocouple temperature. Several correlations were seen between total dose, tumor growth delay, percent skin necrosis, and temperature at the second thermocouple after several superficial and interstitial treatments. Statistically significant improvements in tumor growth delay (at 42 and 45 degrees C) and increased percent skin necrosis at all temperatures were observed after superficial versus interstitial treatment.


Interstitial photoradiation injury of normal brain. The effect on normal brain of continuous interstitial laser irradiation at 630 nm through an implanted cylindrical-shape, diffusion-tipped optical fiber was studied in the rat. Brain water content in the laser irradiation area (LIA) and Evans blue (EB) dye content in selected areas of the brain were measured for different laser power outputs from 0 to 250 mW after 5 minutes of photoradiation. The degree and nature of tissue damage was examined histologically and correlated with the laser power level. There is significant brain damage, blood brain barrier (BBB) disruption, and brain edema in LIA for laser power outputs in excess of 100 mW from the diffusion tip (p less than 0.001). Brain edema in the LIA is strongly correlated with BBB disruption indicated by the presence of EB. Histologically, the cortical surface was more susceptible than deeper white matter regions to interstitial laser irradiation. Possible indirect mechanisms of brain injury from interstitial laser irradiation are discussed.


Therapeutic effect of Ga-Al-As diode laser irradiation on experimentally induced inflammation in rats. We produced experimental inflammation models in rats by carrageenin and studied the effect of Ga-Al-As diode laser irradiation (780 nm, continuous wave, 31.8 j/sec/cm2, spot size of 0.2 mm) on inflamed regions compared with those of indomethacin, a potent anti-inflammatory agent. We found that a low-power infrared laser has an anti-inflammatory effect on carrageenin inflammation. A low-power laser inhibits: (1) the increase of vascular permeability during the occurrence of an acute inflammation in the carrageenin-air-pouch model, (2) edema in the acute stage in the carrageenin-paw-edema model, and (3) the granuloma formation in the carrageenin-granuloma model after receiving laser irradiation once daily. In all cases, irradiation for less than 10 min was sufficient to inhibit the inflammation by 20-30%. The inhibitory effect of laser irradiation was not comparable to that of indomethacin (4 mg/kg, i.o.) in the air-pouch model and the paw-edema model, whereas laser irradiation was more potent than that of daily administration of indomethacin (1 mg/kg, i.o.) in the granuloma model. In future studies of the mechanism of laser effect, it should be noted that irradiating a rat twice, before and after the provocation of inflammation, was essential in order to achieve an effective inhibition of paw-edema.


Dye-mediated bactericidal effect of He-Ne laser irradiation on oral microorganisms. Little attention has been given to the bactericidal effect of laser irradiation, particularly using low-power energy lasers. It has been demonstrated that He-Ne laser light has an inhibitory action on dental plaque. The purpose of this study was to investigate the bactericidal effect of He-Ne laser irradiation on cariogenic microorganisms. The bactericidal effect was determined by the formation of a growth-inhibitory zone or by the counting of viable bacterial colonies. Streptococcus sobrinus AHT that is a Gram-positive microorganism was sensitive to He-Ne laser light, but Escherichia coli, a Gram-negative microorganism, was resistant. The effect of several dyes necessary to instigate a bactericidal action was also examined. A growth-inhibitory zone was observed using 10 kinds of blue, purple, or green dyes, which were mainly phenylmethane dyes. The leakage of potassium from S. sobrinus AHT following laser irradiation was determined using an atomic absorption spectrophotometer. The leakage began to increase following irradiation for 2 min, and reached a plateau following irradiation for 30-60 min. Moreover, to examine some changes in the dye itself following laser irradiation in the absence of bacteria, ultraviolet-visible absorption spectra and 1H NMR spectra were recorded. In this study, it was indicated that the bactericidal effect on cariogenic bacteria by He-Ne laser irradiation was efficient only in the presence of specific dyes. It is suggested that this laser may be suitable for clinical applications in preventive dentistry.


Role of immersion refractometry for investigating laser-induced effects in cells. The broad background of scattered light observed in spectra of cell suspensions is reduced by factors of up to 20 by immersion refractometry allowing for improved spectroscopic determination of the absorption properties of cells in the 325-820 nm range. Refractive-index matched spectra of E. coli C1a exhibit a set of resonant features near 422, 561, and 582 nm. Exposure wavelengths are chosen based on this spectrum and cell viability is investigated in E. coli suspensions exposed to 350, 400, 422, 440, and 700 nm radiation delivered in nanosecond pulses with total doses from 500 millijoules to 60 Joules. We observe a loss in cell viability for doses greater than 1 Joule at 422 nm and for all doses at other wavelengths; exposures of less than 1 Joule at 422 nm enhance growth. Excluding exposures at wavelengths within the resonant feature, longer wavelengths are less effective at reducing the viability of E. coli C1a. This indicates the occurrence of at least two absorption processes.


Forearm flap in orthopaedic and hand surgery. This study was undertaken in order to evaluate the usefulness of the forearm flap in reconstruction of severe injuries of the upper and lower extremities. A total of 34 patients with extensive skin defects of the upper and lower extremities were treated using radial forearm flaps during the last 4 years. Twenty-four patients had lower extremity injuries, while the remaining ten had upper extremity reconstruction. In two patients, the radial forearm flap was used as an island flap in retrograde direction for coverage of skin defects of the dorsum of the hand. In two patients, the palmaris longus tendon was included in the flap to reconstruct the extensor tendon of the index finger. In one patient, it was used as an innervated flap to cover a skin neurotrophic defect of the sole of the foot. None of the patients had the bony portion of the radius included. Of the 34 flaps, 29 survived. Of the remaining 5, 3 failed totally and 2 partially. The 2 partially failed flaps required reoperation and revision of the venous anastomosis, which was found to be occluded. The patients, eight women and twenty-six men, were examined and questioned regarding the cosmetic appearance of the donor site. The final cosmetic appearance was acceptable to all patients. We conclude that the radial forearm flap is a useful, easily elevated flap, suitable for skin defects of upper and lower extremities. Major advantages of the flap are its ability to be used as an island flap and that the surgery can be performed under axillary block anaesthesia.


Results after repair of traumatic arterial damage in the forearm. Eighty-three patients, with 96 instances of radial or ulnar arterial damage, were treated over a four-year period using microsurgical techniques. Thirty-one patients, with 43 arterial repairs, were available for evaluation. In the 14 patients with a single arterial repair, 4 resulted in thrombosis (71.5% success rate). When both arteries required repair, our success rate was 75.9%, with 7 thromboses. The overall success rate was 74.5%. We attribute the high patency rate to strict microsurgical technique, the repair of the vessels in the early stages after trauma, and the use of vein grafts after complete resection of the damaged portion of the vessel.


Clinical analysis and evaluation of the function of replanted and revascularized parts of the upper limb. During the past four years, we have attempted to replant and revascularize 57 hands and fingers in 43 patients. The total success rate was 81%. Replantation of hands severed cleanly (guillotine type) and those with slight local crush injuries had the highest success rate. As for hand function, amputations at zones III and IV with end-to-end suturing of the tendons and nerves presented the highest degree of function when evaluated objectively and subjectively. All patients presented a high degree of satisfaction with the outcome of the operation.


Free vascularized fibular graft: a versatile graft for reconstruction of large skeletal defects and revascularization of necrotic bone. The microvascular transfer of a fibular graft that was initially introduced for the treatment of massive bone loss in the appendicular skeleton has more recently found another useful application in the management of the osteonecrosis of the femoral head. Since 1989, we have successfully used the free vascularized fibula in two patients with segmental bone loss, one in the forearm and the other in the distal femur. We have also attempted revascularization of the femoral head in fifteen patients (eighteen hips) with osteonecrosis. Although our follow-up is short, the results have been encouraging and this method is now the treatment of choice in the early stages of the disease.


Use of free flaps in the aesthetic reconstruction of face and neck deformities. Three cases of face and neck deformities, reconstructed with free flaps in order to obtain better aesthetic results, are presented here. Nasal tip, cartilage, and soft tissue defects as well as facial burn contractures were reconstructed with a free radial forearm flap, latissimus dorsi myocutaneous flap, and groin flap, respectively. Case specifics are discussed.


Post-discectomy perineural fibrosis: comparison of conventional versus microsurgical techniques. The lumbar spines of twenty-one dogs were used as an experimental model. The animals were divided into three groups. In the first group, selective damage to the perimeningeal blood vessels was induced and the resultant hematoma was left untouched in the spinal canal. In the second group, the posterior longitudinal ligament was incised, and in the third group, the posterior longitudinal ligament was incised and damage induced to the perimeningeal blood vessels. The pathology examination revealed: (1) the hematoma itself did not lead to the formation of perineural fibrosis, (2) the incision of the posterior longitudinal ligament led to the formation of a limited amount of fibrosis, and (3) the coexistence of hematoma and incision of the posterior longitudinal ligament led to the formation of extensive perineural fibrosis. When discs are removed using microsurgical techniques, it is possible to avoid the formation of the postoperative hematoma and consequently to eliminate the perineural fibrosis.


Experimental evaluation of the length of microvenous grafts under normal tension. Four groups of microvenous grafts of 2, 4, 6, and 10 cm were used to determine whether length under normal tension affects patency rates in bridging arterial defects. Tension was evaluated by allowing the grafts to assume their normal orientation and length by filling them with blood, under physiological pressure, after the completion of the proximal anastomosis. The length was estimated through the normal range of motion such that the graft would not be too loose and/or tortuous following final anastomosis. The patency rates in the four length groups of this study ranged between 80% and 83%, with no statistical difference between groups. No statistically significant correlation was found between graft length and patency rate. It was concluded that the length of venous autografts does not affect patency.


Hemodynamic and histological differences in end-to-side anastomoses. An end-to-side anastomosis is often a necessity in replantation surgery. This study examines the effects of elliptical versus slit arteriotomy and the angle of anastomosis on vessel patency and aneurysm formation. Forty male rats were divided into four groups of ten rats each. The right and left common iliac arteries were selected as a model. The results showed no significant statistical difference in patency rates among our four experimental groups. The end-to-side anastomotic technique of choice uses the elliptical arteriotomy placed at a 90 degrees angle. It provides good visualization and easy suture placement.


Cutaneous trunci myocutaneous flap: a new and reliable model for muscle free flap research in the rat. This paper deals with the first myocutaneous flap in the rat. It is located just caudal to the axilla and consists of the insertion of the cutaneous trunci muscle with overlying subcuticular fat and skin and is supplied by the lateral thoracic vessels. It exhibits an all-or-none survival pattern. Because the skin can serve as a marker for muscle viability, we believe that this flap has great potential in future free muscle research.


Free flap transfer of the cutaneous maximus muscle in the rat: comparison to the latissimus dorsi muscle flap. A new rat model of free muscle flap transfer is presented. Microvascular transplantation of the cutaneous maximus muscle flap is performed at the groin site, with anastomosis of the axillary vessels to the appropriate femoral vessels. This muscle flap has many useful attributes for experimental manipulation. It has a high success rate following transplantation, the anatomy is consistent, the dissection is straightforward, the length of pedicle is relatively long (10 mm), the vessels for repair are of sufficient size (1.0-1.35 mm diameter), and the microsurgical procedure can be performed in a relatively short period. The donor site deficit causes minimal impairment to animal mobility, and no evidence of limb ischemia is noted after ligation of the axillary vessels. The cutaneous area adjacent to the muscle is perfused by muscular perforators supplied by the flap pedicle; thus a skin island may be used to monitor the flap or to create a composite myocutaneous transfer. The cutaneous maximus muscle has mixed muscle types and anatomic dimensions similar to those of the latissimus dorsi muscle, and it provides ample tissue for pharmacological and biochemical studies, yet it presents easier dissection and microanastomoses than the latissimus flap, with more potential for versatility in application. The advantages of this muscle flap make it a very useful experimental model for flap transfer research.


Prefabrication of free flaps using the omentum in rats. The omentum used as a vascular carrier is a new experimental model for prefabrication of free flaps in the rat. A 2.5 x 4 cm patch of omentum with gastroepiploic vessels and its rich vascular arcades was transferred under a bipedicled 2.5 x 6 cm right abdominal panniculocutaneous flap. At subsequent time intervals (postoperative days 0, 3, 5, 7, and 14), the skin pedicles were divided and the skin flap was raised as a composite island flap vascularized only by the underlying omental patch. The composite flap was then sutured back in place. Prefabricated flaps examined postoperatively demonstrated a dye fluorescence index (DFI) of 4.60% +/- 2.82% and 1.67% +/- 4.08% flap survival (FS) in the day 0 group; in the day 3 group, 9.83% +/- 1.47% of DFI and 15.00% +/- 10.35% of FS; in the day 5 group, 28.39% +/- 5.25% of DFI and 86.0% +/- 12.74% of FS (P less than 0.05); in the day 7 group, 38.19% +/- 7.52% of DFI and 98.13% +/- 3.72% of FS (P less than 0.05); and, in the day 14 group, 49.55% +/- 6.70% of DFI and 100% of FS (P less than 0.05). India-ink injection and histologic examination confirmed revascularization of the overlying skin by day 5.


Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation. Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with [alpha-32P]-GTP. Four of the proteins were partially purified and the N-terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity to Bacillus stearothermophilus triosephosphate isomerase, two were 100% identical to the predicted sequences of the B. subtilis ptsI and ptsH genes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown.


Co-ordinate expression of virulence genes during swarm-cell differentiation and population migration of Proteus mirabilis. The uropathogenic Gram-negative bacterium Proteus mirabilis exhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multinucleate, hyperflagellate swarm cells capable of rapid and co-ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysin and metalloprotease, which are believed to be central to the pathogenicity of P. mirabilis. In addition, the ability of P. mirabilis to invade human urothelial cells in vitro was primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non-flagellated, nonmotile transposon insertion mutants were unable to invade urothelial cells and they generated only low-level activities of haemolysin, urease and protease (0-10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild-type activities of haemolysin, urease and protease, but their ability to enter urothelial cells was three- to 10-fold lower.(ABSTRACT TRUNCATED AT 250 WORDS)


The role of the pro-sequence in the processing and secretion of the thermolysin-like neutral protease from Bacillus cereus. The Bacillus cereus cnp gene coding for the thermolysin-like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre-pro-protein (M(r) = 61,000); the pro-peptide is approximately two-thirds of the size of the mature protein. The pro-sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro-sequences are shown to be homologous to the pro-sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro-protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro-sequences. The deletion results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N-terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro-sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro-protein.


icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. We have characterized the icsB gene which is located on the virulence plasmid pWR100. After inactivation of icsB, the mutant strain remained invasive, but formed abnormally small plaques on HeLa cell monolayers, colonized only the peripheral cells of Caco-2 islets, and was unable to provoke a keratoconjunctivitis in guinea-pigs. Examination of infected HeLa cells showed that the icsB mutant was able to lyse the phagocytic vacuole and to form protrusions at the surface of infected cells, but, unlike the wild type, remained trapped in protrusions surrounded by two membranes. These results indicate that IcsB is involved in the lysis of the protrusions, a step necessary for intercellular spread.


Activator-independent formation of a closed complex between sigma 54-holoenzyme and nifH and nifU promoters of Klebsiella pneumoniae. The alternative sigma factor sigma 54 is required for transcription of nitrogen fixation genes in Klebsiella pneumoniae and other diazotrophs. The nif genes, and other E sigma 54-dependent genes whose products are necessary for a wide range of processes, are postively regulated. A unifying model that is well supported by studies on nif and other nitrogen-regulated (ntr) genes includes the central tenet that sigma 54 confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator-independent complex between E sigma 54 and the NifA-dependent K. pneumoniae nifH and nifU promoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the E sigma 54 recognition sequence.


A Salmonella dublin virulence plasmid locus that affects bacterial growth under nutrient-limited conditions. This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal-medium containing various sole carbon/energy sources, unlike the wild-type and plasmid-cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the phenotypic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal-medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.


In vivo characterization of tus gene expression in Escherichia coli. The tus gene encodes a DNA-binding protein (Tus) that inhibits replication forks at specific block-sites within the terminus region of the Escherichia coli chromosome. One of these block-sites, TerB, is adjacent to the tus gene. Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregulates expression at this weak promoter. We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames. This readthrough transcription into tus is reduced in the presence of Tus protein.


An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity. Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life-threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase-chain-reaction-based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS-polyacrylamide gel electrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild-type cells, the mutant had undetectable activity on azocollagen and elastin-Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.


Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases. The nucleotide sequences of blaLCR-1 and blaOXA-5 beta-lactamase genes have been determined. Polypeptide products of 260 and 267 amino acids with estimated molecular masses of 27 120 Da and 27,387 Da were obtained for the mature form of LCR-1 and OXA-5 proteins. A progressive alignment was used to evaluate the extent of identity between LCR-1 and OXA-5 with 29 other beta-lactamase amino acid sequences. The data showed that both belong to class D. We identified amino acids conserved in 24 positions for class A beta-lactamases and in 28 positions for five class D enzymes. The structural similarities between class A and class D beta-lactamases are more extensive than indicated by earlier biochemical studies with overall 16% identity between both classes. From the alignment, dendograms were constructed with a distance-matrix and parsimony methods which defined three major groups of proteins subdivided into clusters giving insight on beta-lactamase phylogeny and evolution.


Prompt comprehension in UNIX command production. We hypothesize that a cognitive analysis based on the construction-integration theory of comprehension (Kintsch, 1988) can predict what is difficult about generating complex composite commands in the UNIX operating system. We provide empirical support for assumptions of the Doane, Kintsch, and Polson (1989, 1990) construction-integration model for generating complex commands in UNIX. We asked users whose UNIX experience varied to produce complex UNIX commands, and then provided help prompts whenever the commands that they produced were erroneous. The help prompts were designed to assist subjects with respect to both the knowledge and the memory processes that our UNIX modeling efforts have suggested are lacking in less expert users. It appears that experts respond to different prompts than do novices. Expert performance is helped by the presentation of abstract information, whereas novice and intermediate performance is modified by presentation of concrete information. Second, while presentation of specific prompts helps less expert subjects, they do not provide sufficient information to obtain correct performance. Our analyses suggest that information about the ordering of commands is required to help the less expert with both knowledge and memory load problems in a manner consistent with skill acquisition theories.


The correlation of feature identification and category judgments in diagnostic radiology. Expert and novice radiologists were given films accompanied by clinical histories that supported a diagnosis either of bronchiolitis or of normal. To provide a plausible task context, some films were radiologically unambiguous and were accompanied by histories consistent with them. For a set of radiologically difficult films from confirmed normal or bronchiolitis patients, fictitious normal or abnormal histories were counterbalanced with the films. The clinical histories affected ratings both of diagnosis and of features present on the difficult films. Thus, uncertainty about individual features evidently was affected by history, and features did not act as an independent source of information. The dependence of feature calls on an overall judgment was also suggested by intra-observer agreement in another study in which an explicit diagnosis was not requested. It is unclear whether the history increased discrimination between normal and abnormal films, or indiscriminately added evidence for or against the disease. Factors are discussed that make it appropriate for feature identification to be partially dependent on category identification.


Memory recall in a process control system: a measure of expertise and display effectiveness. Previous research has shown that memory-recall performance is correlated with domain expertise. In this study, a process control system was selected as a vehicle for conducting research on memory recall. The primary purposes of the present work were to determine if the classic expertise effects originally obtained in chess generalize to this novel domain and to evaluate the validity of memory recall as a measure of display effectiveness. Experts and novices viewed dynamic event sequences showing the behavior of a thermal-hydraulic system with two different displays, one that only contained information about the physical components in the system (P) and another that also contained information about higher order functional variables (P+F). There were three types of trials: normal, where the system was operating correctly; fault, where a single fault was introduced; and random, where the system's behavior did not obey physical laws. On each trial, subjects were asked to recall the final state of the system and to diagnose the system state. The P+F display resulted in superior diagnosis performance compared with the P display. With regard to memory, there was some evidence of an interaction between trial type and expertise, with experts outperforming novices but primarily on meaningful trials. In addition, memory for the subset of variables most critical to diagnosis was better with the P+F display than with the P display, thereby indicating that memory recall can be a sensitive measure of display effectiveness. The results also clarify a theoretical problem that has existed for some time in the literature, namely, the conditions under which expertise advantages are to be expected in memory-recall tasks. Collectively, these findings point to the potential benefits of adopting an applied context as a test bed for basic research issues.


Importance of the kind of cue for judgments of learning (JOL) and the delayed-JOL effect. The delayed-JOL effect is the finding in which judgments of learning (JOLs) are more accurate at predicting eventual recall when they are made a short time after study rather than immediately after study. The present research replicated this effect and found that the kind of cue that is used for JOLs is critical. In particular, following the study of stimulus-response paired associates, there is an extremely robust delayed-JOL effect when the cue for JOLs is the stimulus alone (every one of 45 subjects showed the effect); however, there is little, if any, delayed-JOL effect when the cue for JOLs is the stimulus-response pair. This finding has important implications for education: To have the greatest accuracy at predicting eventual recall, a person should make JOLs not immediately after study but, instead, shortly after study (i.e., delayed JOLs) with the cue for JOLs being the stimulus alone. The theoretical mechanisms for the delayed-JOL effect are currently unknown, but some speculations are offered.


The methodology of testing naive beliefs in the physics classroom. Many undergraduates harbor a variety of misbeliefs about physical objects in motion--for instance, that a bomb will fall straight down when dropped from a moving airplane. The evidence that these misbeliefs are resistant to correction by college-level physics courses, however, has often been based on methodologies that lack adequate internal validity. We used a quasi-experimental "before and after" design to assess the impact of two college physics courses, and we examined selection-bias, test-retest, and task-format factors directly. Initial accuracy and significant improvements due to instruction varied considerably by problem category and subject group; thus, in several ways, the results refute the general conclusion that conventional physics instruction does little to correct students' misbeliefs. We conclude by advocating the quasi-experimental approach for studies of naive beliefs in physics as well as for other situations in which the impact of classroom instruction is of interest.


Information selection and use in hypothesis testing: what is a good question, and what is a good answer? The process of hypothesis testing entails both information selection (asking questions) and information use (drawing inferences from the answers to those questions). We demonstrate that although subjects may be sensitive to diagnosticity in choosing which questions to ask, they are insufficiently sensitive to the fact that different answers to the same question can have very different diagnosticities. This can lead subjects to overestimate or underestimate the information in the answers they receive. This phenomenon is demonstrated in two experiments using different kinds of inferences (category membership of individuals and composition of sampled populations). In combination with certain information-gathering tendencies, demonstrated in a third experiment, insensitivity to answer diagnosticity can contribute to a tendency toward preservation of the initial hypothesis. Results such as these illustrate the importance of viewing hypothesis-testing behavior as an interactive, multistage process that includes selecting questions, interpreting data, and drawing inferences.


The role of availability in the estimation of national populations. In two parallel experiments, conducted 17 months apart, we examined the relations among population estimates, availability, and media coverage of the 100 countries with populations of 4 million or more. The results were consistent with the main hypothesis, that availability influences population estimates. Specifically, we found that (1) rated knowledge of each country (our measure of availability) was strongly correlated with estimated population for that country; (2) rated knowledge was more strongly correlated with estimated population than were other plausible predictors, such as land area; (3) a strong partial correlation between rated knowledge and estimated population remained even after true population and true land area were partialed out; and (4) longitudinal changes in rated knowledge predicted longitudinal changes in estimated population. Also as hypothesized, media coverage was positively related to availability and to population estimates. Further, we found that population estimates, rated knowledge, and amount of media coverage that each country received were very stable over the year-and-a-half period. These results led us to consider when availability is most likely to be relied upon heavily in quantitative estimation and how it may be integrated with other knowledge to derive quantitative estimates.


Working-memory mediation of adult age differences in integrative reasoning. Three research methods were used to investigate the hypothesized mediational influence of working memory on age-related differences in integrative reasoning. Results from all three procedures were consistent with the hypothesis because (1) statistical control of an index of working memory attenuated the age differences in reasoning accuracy, (2) young adults were more accurate than older adults in a measure reflecting the preservation of information during processing, and (3) young adults performing the task with a concurrent memory load exhibited a qualitative pattern of performance similar to that of older adults performing the task without a concurrent memory load.


The effect of foregrounding on readers' use of predictive inferences. This research extends previous attempts to determine whether subjects make predictive inferences during comprehension. For example, when subjects read a passage about someone falling out of a 14th-story window, do they infer that the person is dead? Previous research in which lexical decision, word naming, and recognition tasks have been used for detecting predictive inferences has had mixed results. In experiment 1, a word-stem completion task was used to test for predictive inferences. The word stems were formed from target inferences that followed either priming or control passages. The data revealed that predictive inferences are generated only about concepts that are foregrounded in the passages. In Experiments 2 and 3, lexical decision and naming were used to test for predictive inferences. The lexical decision data replicated the word-stem completion data. A control experiment ruled out a simple context-checking explanation for the lexical decision results. The naming data indicated that this tasks was not sensitive to elaborative inference generation. The results show that readers make predictive inferences, but do so selectively.


Caricature and face recognition.

Caricature and face recognition. Although caricatures are often gross distortions of faces, they frequently appear to be super-portraits capable of eliciting recognition better than veridical depictions. This may occur because faces are encoded as distinctive feature deviations from a prototype. The exaggeration of these deviations in a caricature may enhance recognition because it emphasizes the features of the face that are encoded. In two experiments, we tested the superportrait hypothesis and the encoding-by-caricature hypothesis. In the first experiment, caricatures were recognized better than faces, and true caricatures of previously seen faces were recognized better than the faces from which the caricatures had been developed. In the second experiment, faces and their caricatures were tachistoscopically presented in a sequential same/different reaction time task. Subjects were slower to distinguish the stimuli when the face preceded its caricature, indicating that caricatures are more similar to the encoded representation of a face than are stimuli in which the distinctive features are deemphasized.


Nonverbal priming in amnesia.

Nonverbal priming in amnesia. In this experiment, we examined whether a group of well-characterized amnesic patients would exhibit normal priming for novel nonverbal materials after a single exposure. Both amnesic patients and normal control subjects studied line figures and were then given a priming test in which they were asked to reproduce both old (studied) and new (unstudied) figures after a brief exposure. The measure of priming was the number of old patterns drawn correctly relative to the number of new patterns drawn correctly. Both subject groups reproduced more old patterns than new patterns, and the effect was similar in the two groups. In contrast, amnesic patients were significantly impaired on a recognition memory test for the items that had been presented. This study contributes to recent evidence that implicit memory can support the rapid acquisition of novel verbal and nonverbal information. Perceptual priming for such material is independent of the structures damaged in amnesia.


Verbal recoding of visual stimuli impairs mental image transformations. Two experiments were carried out to test the hypothesis that verbal recoding of visual stimuli in short-term memory influences long-term memory encoding and impairs subsequent mental image operations. Easy and difficult-to-name stimuli were used. When rotated 90 degrees counterclockwise, each stimulus revealed a new pattern consisting of two capital letters joined together. In both experiments, subjects first learned a short series of stimuli and were then asked to rotate mental images of the stimuli in order to detect the hidden letters. In Experiment 1, articulatory suppression was used to prevent subjects from subvocal rehearsal when learning the stimuli, whereas in Experiment 2, verbal labels were presented with each stimulus during learning to encourage a reliance on the verbal code. As predicted, performance in the imagery task was significantly improved by suppression when the stimuli were easy to name (Experiment 1) but was severely disrupted by labeling when the stimuli were difficult to name (Experiment 2). We concluded that verbal recoding of stimuli in short-term memory during learning disrupts the ability to generate veridical mental images from long-term memory.


Use of N-([1-14C]hexanoyl)-D-erythro-sphingolipids to assay sphingolipid metabolism. An advantage of using N-([1-14C]hexanoyl)sphingolipids to assay sphingolipid metabolism is their ability to rapidly and spontaneously transfer into biological membranes without destroying membrane integrity. This property allows analysis of the activity of enzymes of sphingolipid metabolism under conditions in which the rate of product formation is not limited by availability of substrate, as is often the case with naturally occurring lipids whose rates of spontaneous transfer are extremely slow. Thus, the use of N-([1-14C]hexanoyl)sphingolipids provides an alternative means for studying sphingolipid metabolism in vitro.


[Correlation between hormonal and metabolic profiles in women with polycystic ovary syndrome]. To investigate the sex hormone status of women with polycystic ovary syndrome (PCO) and to relate this to serum levels of glucose, insulin, lipids and lipoproteins, 90 women with PCO (30 obese: BMI greater than 30 kg/m2; 30 overweight: BMI greater than 25- less than 30 kg/m2; 30 non obese: BMI less than 25 kg/m2) and 60 normal ovulatory women (20 obese; 20 overweight; 20 non obese) were studied. The women with PCO had significantly increased LH, FSH and androgen levels and significantly decreased SHBG levels compared to the normal women. Obese women with PCO had higher concentrations of fasting glucose, fasting insulin, incremental glucose area, incremental insulin area and lipid than overweight and non obese women with PCO and overweight and non obese control subjects, but were similar in obese normal women. There were decreases in high-density lipoproteins levels in both the obese groups (obese PCO and obese control women). Lipid and lipoprotein concentrations did not differ in the obese, overweight and non obese PCO women compared to the normal groups while HDL cholesterol were decreased in obese PCO and obese control women. The correlations between hormone, glucose, insulin, lipid and lipoprotein levels were different among the six groups. Non obese PCO women had: inverse correlations between free testosterone and incremental glucose area (r = -0.5128, P = 0.03); positive correlations between SHBG and alpha-lipoproteins (r = 0.9159, P = 0.001). Non obese normal women had: positive correlations between fasting insulin and total testosterone (r = 0.5272, P = 0.043) and between SHBG and beta-lipoproteins (r = 0.7445, P = 0.014) and LDL cholesterol (r = 0.7360, P = 0.010).(ABSTRACT TRUNCATED AT 250 WORDS)


[Profile of thyroid hormones in aging: evaluation of a hospitalized elderly population]. The aim of the study is to evaluate thyroid serum levels in a geriatric community to confirm the presence of a low T3 syndrome during normal ageing. The authors consider 413 subjects (125 male and 288 female) admitted to our Geriatric Division. The group affected by thyroid and extrathyroid disease (such us malnutrition, diabetes mellitus, renal failure, etc.) was withdrawn. In the selected patients (271) was operated a statistical evaluation to correlate the hormonal parameters (T3, T4, TSH, FT3, FT4) with age and sex. According to international literature, we confirm a progressive T4 and FT4 reduction (p less than 0.05) during ageing, both in male and in female. These data range within normal values. On the contrary, TSH shows no modifications with age and sex. Unlike all other parameters, T3 presents a more evident decrement with age, confirming a low T3 syndrome.


[Effect of treatment with biosynthetic GH on thyroid function in patients with an isolated deficiency of GH]. The use of GH treatment in subjects with a GH deficiency has led to contrasting results concerning its impact to develop thyroid hyperfunction, whereas many others have underlined the possible onset of hypothyroidism. A number of studies have been carried out over short periods in subjects with multiple tropin deficiencies, in healthy adults or adults with GH deficiencies, in healthy adults or adults with GH deficiencies. The aim of the present study was to assess the effect of prolonged treatment with biosynthetic GH on thyroid function in children with an isolated idiopathic GH deficiency. The study included 8 children (mean age 10.4 +/- 0.8 years) with GH deficiencies treated with biosynthetic GH and 8 children with familial retarded stature of a similar age (mean age 10.3 +/- 0.7 years) who represented the control group. Serum levels of T3, T4, FT3, FT4 and TSH were measured at the start of the study and after one year of continuous GH treatment in subjects with GH deficiency; the same tests were performed in the control group on recruitment and after one year's observation without therapy. T4 and FT4 levels diminished, but not significantly, whereas there was a significant increase in plasma levels of T3 and FT3 (p less than 0.01); TSH values were significantly reduced in the treated group (p = 0.025). No significant variations in thyroid parameters were found in the control group. These data support the hypothesis of an increased peripheral conversion of T4 into T3 due to GH therapy; in conclusion, however, no significant variation in thyroid function was observed following GH replacement therapy, even if prolonged, in subjects with an idiopathic isolated GH deficiency.


[Beta-2-microglobulin in hyperthyroidism]. The aims of the study were to clarify the cause of increased serum beta 2-microglobulin as a marker of thyroid hyperfunction. Serum beta 2-microglobulin was measured in 31 untreated hyperthyroid patients, all of them with normal renal function. Twenty-one subjects were affected by diffuse toxic goiter and 10 by toxic adenoma. Serum free thyroid hormones, TSH, anti-thyroglobulin and anti-microsomal antigen autoantibodies were determined, too. Thyroid hormone and creatinine levels did not differed between both sets of patients. beta 2-microglobulin was higher than normal in 90% of cases with diffuse toxic goiter and in 70% of those with toxic adenoma (p less than 0.05), but mean beta 2-microglobulin concentrations didn't differ between the two groups. No difference was found in beta 2-microglobulin levels in diffuse toxic goiter group according to the presence or absence of autoantibodies. beta 2-Microglobulin and thyroid hormones were not correlated in either diffuse toxic goiter and toxic adenoma groups. These data confirm the high prevalence of elevated beta 2-microglobulin concentrations in hyperthyroidism. As renal function was normal, this rise is due to beta 2-microglobulin overproduction. This increased production is a hormone mediated effect, even if lymphocyte activation may contribute in diffuse toxic goiter. beta 2-microglobulin is not correlated with thyroid hormone concentrations so that at present it isn't a useful marker of hyperthyroidism severity for practical purposes.


[Clinical evaluation of desmopressin (DDAVP) in diabetes insipidus: solution vs tablets]. Efficacy and tolerability of perorally administered desmopressin were evaluated in 12 adult patients suffering from central diabetes insipidus. According to the results of the study desmopressin should be administered at a higher dosage than 300 micrograms/die to obtain the same antidiuretic effect as with the intranasal administration. No significant changes in body weight, arterial pressure and blood chemistry parameters have been detected over the whole administration period and only some slight side-effects occurred. Oral desmopressin may be considered as an effective alternative to traditional intranasal administration and it may significantly improve patients' compliance.


[Effect of octreotide on blood glucose levels in poorly compensated insulin-dependent diabetics on insulin treatment]. The metabolic effects of a long-acting somatostatin analogue, octreotide, in type I diabetic patients on conventional insulin therapy have been evaluated. Octreotide has been administered by subcutaneous continuous infusion employing a minipump; in 10 patients the infusion was performed from 08.00 a.m. to 08.00 p.m. and in 7 patients from 08.00 p.m. to 08.00 a.m. In both groups the drug dose was 50 mcg/12h and before starting the infusion an additional dose of 12.5 mg was rapidly injected s.c. In a third group of 8 patients octreotide was administered at a dosage of 50 mcg by subcutaneous injections at 07.00 a.m.; 3.00 p.m.; 11.00 p.m. A significant reduction of glucose levels was obtained in all patients and the results observed by minipump or by multiple injections were superimposable. It is to note that the daily drug dose employed by minipump was smaller. The evaluation of octreotide usefulness, in addition to conventional insulin therapy, in the management of type I diabetic patients, needs further extensive studies.


Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model. Gene conversion--apparently non-reciprocal transfer of sequence information between homologous DNA sequences--has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo+ gene by homologous recombination. We found that all the neo+ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo+ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec+ strain or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec+ strain, led us to propose a mechanism for this biased gene conversion. This "successive half crossing-over model" proposes that the elementary recombinational process is half crossing-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.


Characterization of the Aspergillus niger pelB gene: structure and regulation of expression. The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.


Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase. A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.


Regulatory sequences for expressing genes in oomycete fungi. Promoter and terminator sequences from a range of species were tested for activity in the oomycetes, a group of lower fungi that bear an uncertain taxonomic affinity to other organisms and in which little is known of the sequences required for transcription. Transient assays, using the reporter gene beta-glucuronidase (GUS), were used to examine the function of these promoters and terminators in the plant pathogens Phytophthora infestans and P. megasperma f. sp. glycinea, and in the saprophytic water mold, Achlya ambisexualis. Oomycete promoters, isolated from the ham34 and hsp70 genes of Bremia lactucae and the actin gene of P. megasperma f. sp. glycinea, resulted in high levels of GUS accumulation in each of the three oomycetes. In contrast, little or no activity was detected when promoters from higher fungi (four ascomycetes and one basidiomycete), plants, and animals were tested. The terminator from the ham34 gene resulted in much higher levels of GUS accumulation than did others, although an oomycete terminator was not absolutely required for expression. Transcript mapping of RNA from stable transformants confirmed accurate initiation from the B. lactucae hsp70 promoter and termination within 3' ham34 sequences in P. infestans. Our results indicate that the transcriptional machinery of the oomycetes differs significantly from that of the higher fungi, but that enough conservation exists within the class to allow vectors developed from one oomycete species to be used for others.


Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli. Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA211 each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein.


New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction. A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.


A topological model for the haemolysin translocator protein HlyD. A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active HlyD-LacZ fusion proteins were only generated when lacZ was fused to hlyD within the first 180 bp (60 amino acids). HlyD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD). Active insertions of phoA into the middle region of hlyD were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)


NPK1, a nonessential protein kinase gene in Saccharomyces cerevisiae with similarity to Aspergillus nidulans nimA. A new protein kinase gene [called NPK1 (for nonessential protein kinase)] has been found on chromosome I of Saccharomyces cerevisiae between CDC15 and ADE1. The 435 amino acid/48 kDa gene product is very similar to known protein kinases. It is most closely related to the nimA protein of Aspergillus nidulans, displaying 45.9% identity and 63.5% similarity in the protein kinase domain. A 1.4 kb transcript of the NPK1 gene was detected. Disruption of the NPK1 gene impedes neither growth on glucose or a variety of other carbon sources, nor mating or sporulation.


The role of promoter elements of a ribosomal protein gene in Saccharomyces cerevisiae under various physiological conditions. Previous work in our laboratory has shown that the 5' nontranscribed promoter region of the gene for ribosomal protein (rp) S16A-1 of Saccharomyces cerevisiae, when fused to a lacZ gene, is necessary and sufficient to cause an increase in expression of the heterologous lacZ gene fusion product after cells have been shifted from a glycerol to glucose carbon source. This increase in expression is characteristic of that observed with the native rp gene. We have sought to define more precisely those areas of the promoter that may be involved in the differential expression/regulation of RPS16A-1 when host cells are subjected to a variety of nutritional environments. It has already been demonstrated by others that the promoter regions of most rp genes contain at least one consensus element, designated UASrpg, which is necessary for the transcriptional activation and maintenance of expression of the gene during steady-state growth in rich media. Our main experimental approach has been to create a series of 5' end deletions in the promoter region of RPS16A-1. The individual truncated promoter fragments were then ligated to a lacZ fusion reporter construct. By assaying the cells for production of beta-galactosidase and determining the abundance of lacZ mRNA, we have been able to determined the extent of fusion product expression. We assayed cells under three physiological conditions: steady-state growth in glucose, steady-state growth in glycerol and during sporulation. We report four main findings of our work.


Structure of the genes encoding Hordeum vulgare (1----3,1----4)-beta-glucanase isoenzymes I and II and functional analysis of their promoters in barley aleurone protoplasts. Barley (1----3,1----4)-beta-glucanase isoenzyme II is synthesized in the aleurone cells during germination and secreted into the endosperm for hydrolysis of the cell walls. Its synthesis is stimulated by gibberellic acid (GA3) and repressed by abscisic acid. The gene for isoenzyme I is expressed in the aleurone, scutellum and prominently in young leaves. Close functional relatedness between the two enzymes is attested by 92% identity at the level of the amino acid sequence. The structural genes for the two enzymes each contain a large intron of 2505 bp and 2952 bp, respectively, in the codon for amino acid 25 of the 28-residue signal peptide. During evolution, homologous regions of the two introns have changed position and orientation. Furthermore, a large palindromic sequence of 327 bp in the 5' end of the intron is present only in the gene for isoenzyme II. In transient expression assays using barley aleurone protoplasts and chloramphenicol acetyl transferase as reporter the promoter of the isoenzyme I gene showed no response to GA3. However, removal of a unique 151 bp region extending from positions -402 to -552 upstream of the TATA box permitted low levels of GA3-induced expression of the reporter gene, suggesting a silencer function for this domain. High levels of GA3-responsive expression were obtained in aleurone protoplasts using the promoter of the gene encoding isoenzyme II. Truncation of this promoter revealed that sequences located within 253 bp upstream from the TATA box are sufficient to direct GA3-stimulated expression. Using the homologous barley aleurone protoplast transfection assay, it was possible to reproduce the in vivo expression characteristics of the genes for the barley (1----3,1----4)-beta-glucanase isoenzymes I and II with reporter gene constructs.


A molecular marker to select for freezing tolerance in Gramineae. We isolated, and expressed in Escherichia coli, a gene (Wcs120) that is strongly induced during cold acclimation of wheat. The gene product was purified and used to produce antibodies. Immunoblotting experiments with the anti-WCS120 antibody identified several cold-induced proteins named FTMs for Freezing Tolerance Markers since they are associated with the development of freezing tolerance. This protein family was found to be coordinately regulated specifically by low temperature, highly hydrophilic, stable to boiling, and to have a pI above 6.5. The accumulation kinetics during the acclimation period indicated a positive correlation with the capacity of each genotype to develop freezing tolerance. Accumulation of the proteins was higher in the freezing-tolerant genotype than in the less tolerant one. In addition, their accumulation was more pronounced in the crown and leaf tissues compared with roots, confirming a relationship to the capacity of the different tissues to develop freezing tolerance. Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals. The antibody did not cross-react with any of the non-cereal species examined. The anti-FTMs antibody represents a potential tool for breeders to select for freezing tolerance traits in the Gramineae.


Directed excision of a transgene from the plant genome. The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, beta-glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALSr), were integrated into the genome of tobacco and Arabidopsis. The ALSr gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALSr gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The beta-glucuronidase gene remained active. The excision efficiency varied in F1 progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox x Cre F1 progeny were chimeric and some F2 progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.


Individual variability in the translational regulation of ribosomal protein synthesis in Xenopus laevis. Ribosomal protein synthesis is regulated by controlling the fraction of mRNA associated with polysomes. It is known that this value changes in different developmental stages during Xenopus embryogenesis or, more generally, with changing cell growth conditions. We present here an analysis of the proportion of mRNA loaded on polysomes, carried out with probes for five different ribosomal proteins on several batches of Xenopus embryos obtained from different individuals. The results obtained indicate the existence of probe-dependent and individual differences, which reflect genetic variations in the cis- and trans-acting regulatory elements responsible for translational regulation. The fraction of ribosomal protein mRNA loaded onto polysomes can be used as an index of an individual's capacity for ribosome production.


Recombinant repair of diverged DNAs: a study of homoeologous chromosomes and mammalian YACs in yeast. Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15-20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms+ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.


Cloning and sequencing of the melB gene encoding the melibiose permease of Salmonella typhimurium LT2. The nucleotide sequence of the melB gene coding for the Na+ (Li+)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52,800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.


Expression of pehA-bla gene fusions in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production. In vitro gene fusions were constructed between the polygalacturonase-encoding pehA gene of the Erwinia carotovora subsp. carotovora (Ecc) strain SCC3193 and the bla gene of pBR322. The gene fusions obtained (75-2, 75-5 and 75-6) encoded hybrid proteins with the entire signal peptide and 70, 260 or 327 amino acids (aa) of the mature 376 aa PehA protein, respectively, fused to the mature part of the periplasmic beta-lactamase. All three hybrid proteins remained cell-bound in Ecc. High-level expression of the longer fusions 75-5 and 75-6 in Ecc led to reduced growth and viability of the cells. This phenotype was utilized to select for spontaneous extragenic mutations restoring normal cell growth. Two classes of regulatory mutants were obtained by this selection. First, mutants impaired in the production of several exoenzymes, including polygalacturonase, were found. These were phenotypically similar to the previously characterized Exp- mutants. Secondly, mutants specifically impaired in the production of polygalacturonase (designated PehR-), but producing and secreting wild-type levels of pectate lyase and cellulase, were obtained. The PehR- mutations were shown to affect transcriptional activation of the pehA gene. Furthermore, the PehR- as well as PehA- mutants exhibited a reduced virulence phenotype suggesting that polygalacturonase is a virulence factor in Ecc.


A flower-specific cDNA encoding a novel thionin in tobacco. We isolated a flower-specific cDNA, FST (flower-specific thionin), which encodes a novel thionin from tobacco. Thionins are basic and cysteine (Cys)-rich, low molecular weight proteins found in many plants. They are believed to play a role in plant defense against pathogens. The central domain of the FST protein shares homology with three gamma-thionins. Like other thionin precursors, the FST protein has an N-terminal domain characteristic of a signal peptide and an acidic C-terminal domain. FST mRNA accumulates specifically in developing flowers and its level drops as flowers mature. Transcripts are present in petals, stamens and pistil but are not detectable in sepals. In situ hybridization revealed that FST mRNA is most abundant in the epidermal cells along the adaxial surface of petals, and in the surface cell layers of the carpel and anther walls. If the FST protein indeed has a protective role in flowers, this pattern of spatial distribution of FST mRNA would appear to maximize this effect on the two internal reproductive whorls. A possible biological role for FST is discussed.


Expression of the gene encoding the PR-like protein PRms in germinating maize embryos. The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with F. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. moniliforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.


Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells. In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)


Structure of a receptor-binding fragment from human luteinizing hormone beta-subunit determined by [1H]- and [15N]nuclear magnetic resonance spectroscopy. The structure of the glycoprotein hormones (LH, CG, FSH, and TSH) and their mechanism of receptor recognition are problems of long-standing interest and speculation. Here we describe the two-dimensional [1H]nuclear magnetic resonance ([1H]NMR) analysis of a linear peptide model for the intercysteine sequence (38-57) from the beta-subunit of human (h) LH. This sequence contains functional determinants for receptor binding and postreceptor activation and is predicted by computer-based modeling to fold as a compact minidomain containing a central amphipathic helix. To test this prediction, an Arg-extended disulfide-free (38-57) analog of enhanced solubility was prepared for complementary circular-dichroic and two-dimensional NMR studies. The linear peptide retains ovarian membrane receptor-binding activity. Although the peptide is not highly structured in aqueous solution, circular-dichroic analysis shows partial alpha-helix formation in a lipophilic medium (50% trifluoroethanol). Complete sequential assignment is obtained in 50% trifluoroethanol based on homonuclear and [15N]edited heteronuclear NMR methods. alpha-Helix-related (i,i + 3) connectivities are observed by nuclear-Overhauser effect spectroscopy that define an amphipathic alpha-helical segment (residues 41-48). Additional long range nuclear-Overhauser effects are observed in the C-terminal region that are consistent with beta-turns involving one or more proline residues; these may serve to reverse the direction of the peptide chain. A nuclear-Overhauser effect contact is identified between residues 38 and 55 at opposite ends of the linear sequence, suggesting that a loop configuration is significantly populated in this solvent system. These results, taken together, characterize elements of ordered structure in the 38-57 peptide, which appear to be distinguishing features of hLH (and the homologous region of hCG). We propose that the structure of this peptide provides a model for the structure of the corresponding region of native hLH in the hormone-receptor complex.


Identification of the thyroid hormone-responsive messenger RNA spot 11 as apolipoprotein-A1 messenger RNA and effects of the hormone on the promoter. The induction of rat hepatic mRNA S11 by L-T3 (T3) is a useful model for studying the mechanisms of thyroid hormone action. Although numerous reports have examined the response of mRNA S11 to various physiological and hormonal manipulations, the role of S11 protein in cellular metabolism remains unknown. In this study we show that mRNA S11 is abundantly expressed and regulated by T3 only in liver and small intestine. High levels of the mRNA are present at birth, but drop sharply between 30-60 days of age. These and other features of the S11 gene product were similar to those of rat apolipoprotein-A1 (Apo-A1). The sequence of S11 cDNA was identical to a portion of the Apo-A1 mRNA, thus confirming identity of the S11 mRNA. To examine whether DNA sequences immediately adjacent to the transcription start site mediate the effects of thyroid hormone, we measured the activity of an Apo-A1 gene fragment, U-1 (-474 to -7) using a transient transfection assay. The activity of the full-length U-1 DNA in HuH-7 hepatoma cells was 2- to 2.5-fold higher in the presence of thyroid hormone. This finding closely matched previous results using the in vitro nuclear run-on assay. Internal deletion of a motif that resembles a thyroid hormone response element from U-1 DNA not only abolished the induction by T3, but suppressed promoter activity by 3- to 4-fold in response to the hormone.(ABSTRACT TRUNCATED AT 250 WORDS)


Glucocorticoid regulation of c-myc promoter utilization in P1798 T-lymphoma cells. Glucocorticoids rapidly inhibit the expression of c-myc mRNA in P1798 lymphoma cells. Statistically significant decreases can be observed within 5-10 min after the addition of glucocorticoids. Although transcription of c-myc decreases within a few hours after dexamethasone is added to P1798 cell cultures, nuclear run-on transcription cannot be used to demonstrate that the very early changes in mRNA abundance reflect corresponding changes in transcriptional activity. An RNase protection assay has been used to measure the abundance and rates of turnover of the two major c-myc transcripts arising from the P1 and P2 initiation sites. The relative rates of synthesis of the c-myc mRNAs (i.e. transcription) can be calculated from such data. The abundance of the P2 transcript exceeds that of P1 mRNA by 3- to 4-fold in midlog phase cells. The turnover rates of the two c-myc mRNAs are essentially identical (0.02 min-1), indicating that the P2 promoter is 3-4 times stronger than P1. This was confirmed by measuring the relative transcriptional activities of templates containing the individual c-myc promoters in P1798 extracts in vitro. The expression of P1 and P2 mRNAs decreases at different rates in glucocorticoid-treated cells. A 50% decrease in the abundance of P1 mRNA occurs within 1 h after the addition of dexamethasone. Expression of P2 mRNA is reduced by 50% within 4 h. However, the turnover rates of the major c-myc transcripts do not change in glucocorticoid-treated cells. The t1/2 values of P1 and P2 mRNAs are about 25-30 min and not different from the turnover rates measured in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Apolipoprotein-E messenger RNA in rat ovary is expressed in theca and interstitial cells and presumptive macrophage, but not in granulosa cells. Apolipoprotein-E (apoE) is a constituent of various lipoproteins and is a ligand for cellular lipoprotein receptors. Unlike most apolipoproteins, apoE is synthesized in peripheral tissues, including those engaged in steroidogenesis. ApoE expression in adrenal cells inhibits cholesterol utilization for steroid synthesis and blocks signal transduction via the protein kinase-A pathway. In cultured ovarian thecal/interstitial cells, exogenous apoE has been shown to inhibit LH-induced androgen synthesis. These findings support a role for apoE as an autocrine or paracrine factor involved in regulating steroidogenesis. In the present study in situ hybridization was used to identify cell types that express apoE mRNA in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with PMSG to stimulate follicular development, and from PMSG-treated rats that were subsequently administered hCG to stimulate ovulation and luteinization. ApoE mRNA was localized to theca and interstitial cells of follicles in animals at all stages of the estrous cycle as well as in immature rats treated with PMSG. ApoE mRNA was not detected in oocytes, cumulus cells, or granulosa cells. High levels of apoE mRNA also were expressed by localized clusters of presumptive macrophages in atretic follicles and degenerating corpora lutea. This complex pattern of expression may indicate that apoE has multiple functions in the rat ovary. ApoE made by theca and interstitial cells may act locally as an autocrine factor to regulate androgen production. ApoE made in atretic follicles and regressing corpora lutea may serve to facilitate local transport and reutilization of lipid released as these structures degenerate.


Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire. To advance our understanding of the human immunoglobulin V lambda germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the V lambda I subgroup. These genes can be divided into three sub-subgroups on the basis of greater than or equal to 93% nucleotide sequence homology and greater than or equal to 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed V lambda I genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed V lambda I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other V lambda I germline genes (Humlv101 and Humlv104) which may interfere with their expression.


New cyclosporin A analogue: synthesis and immunosuppressive activity. A synthetic analogue of cyclosporine A, in which an unusual amino acid (4R)-N-methyl-4-butenyl-4-methyl-L-threonine (MeBmt) is replaced with L-threonine (Thr), was synthesized by the solid phase method. Its activity in the humoral response to sheep red blood cells in vitro and in vivo in mice was practically the same as that of cyclosporine A used as a standard, whereas the analogue studied exerted a significantly stronger effect in the delayed type hypersensitivity to sheep red blood cells in mice.


Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis. Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11-33), the loop between the first disulfide bond and helix 2 (residues 54-74), the beginning of helix 3 (residues 109-112), the carboxyl half of helix 4 (residues 167-182), and the final carboxyl terminus segment of the molecule (residues 184-191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54-74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.


Influenza basic polymerase 2 peptides are recognized by influenza nucleoprotein-specific cytotoxic T lymphocytes. Cytotoxic T lymphocytes (CTL) play an important role in limiting viral infections and in eradicating virus from host tissues. Recent progress in understanding the processing and presentation of viral antigens to CTL indicates that the CTL antigen receptor recognizes peptides derived from viral proteins that are bound to an antigen binding groove present in class I major histocompatibility complex (MHC) molecules. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccinia recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that while screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.


Possible use of similar framework region amino acid sequences between human and mouse immunoglobulins for humanizing mouse antibodies. We have previously noted that a specific amino acid sequence could form the second framework region of human, mouse and rabbit immunoglobulin light chains, suggesting that this sequence has been preserved for 80 million years. Through divergent evolution, each species has acquired a different set of framework region sequences; however, these sets still share a few similar or identical amino acid sequences. In the present study, we have identified such sequences for all four framework regions between human and mouse immunoglobulin light and heavy chains. They may be useful in humanizing or reshaping mouse or rat antibodies for therapeutic applications in human patients.


The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes. The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.


AAEM minimonograph #39: digital filtering: basic concepts and application to evoked potentials. Filtering of evoked potentials has been performed in clinical laboratories using both analog and digital methods. Analog methods introduce distortion caused by nonlinear phase shift which may be quite severe. Digital methods, while avoiding distortion caused by phase shift, reveal evoked potential components which may or may not correspond to distinct singular neuroanatomic generators or homogeneous neuroanatomic systems. Thus, components identified with zero phase shift digital filters at restricted bandpass must be compared with components seen in open bandpass recordings. In some specific circumstances, high-pass filtering of short-latency somatosensory-evoked potentials may distinguish slow asynchronous synaptic activity from fast and synchronous synaptic, lemniscal, or axonal activity.