Exercise capacity and breathing mechanics in patients with airflow limitation.

To investigate the impact of expiratory airflow limitation on ventilation during exercise, we studied six control subjects with normal lung function (FEV1/FVC = 79 +/- 6%) and eight patients with borderline-to-mild airflow limitation (FEV1/FVC = 68 +/- 4%) during cycle ergometry. VO2, HR, and VE/MVV were not different between the control subjects or patients during maximal or submaximal exercise. In contrast, five of the eight patients achieved maximal expiratory flow over a large portion (37%) of their tidal volume (VT) during submaximal exercise, whereas none of the control subjects achieved maximal expiratory flow. To estimate the fraction of expiratory capacity used by the control subjects and the patients, we calculated a mechanical ventilatory maximum (VEmaxCal) for each level of exercise using the individual's VT, end-expiratory lung volume (EELV), and maximal expiratory flow-volume curve. The patients used a greater fraction of their VEmaxCal at each level of submaximal exercise (P less than 0.03). Despite the flow limitation during submaximal exercise, EELV was similar between the control subjects and patients. In conclusion, even patients with borderline-to-mild airflow limitation achieve maximal expiratory flow during submaximal exercise and these restrictions are not reflected by VE/MVV nor by EELV.


Substrate utilization during treadmill running in prepubertal girls and women.

Ten prepubertal girls (9.1 yr) and 10 women (24.4 yr) were studied during 30-min runs at the same relative (70% VO2max) (RI) and absolute (7.2 km.h-1) (AI) intensities. Oxygen consumption and respiratory exchange ratio (RER) were monitored continuously during the exercise test. Venous blood samples were obtained before and immediately after exercise and were analyzed for glucose, lactate, FFA, and glycerol. During exercise at the same RI, RER was significantly (P less than 0.05) lower in the girls compared with the women. In addition, RER decreased significantly during exercise in the girls while the change did not reach significance in the women. Lactate concentration increased significantly during the exercise tests, this increase being greater in the women than in the girls during exercise at the same RI, but greater in the girls than in the women during exercise at the same AI. FFA and glycerol concentrations were significantly higher after the exercise tests in both groups. Glucose concentration did not change significantly during the tests in either group. Prepubertal girls rely more on fat utilization and less on carbohydrate metabolism than women during exercise of moderately heavy intensity.


The effect of an anti-G suit on cardiovascular responses to exercise in persons with paraplegia.

The purpose of this study was to determine whether external pressure on legs and abdomen could prevent venous blood pooling in persons with paraplegia and thus positively affect their cardiovascular responses to arm exercise. To investigate this, five male subjects with paraplegia (P), with complete lesions between T6 and T12, and five male control subjects who were wheelchair bound (C) (due to a chronic lower extremity disability), performed submaximal arm-cranking exercise at 20%, 40%, and 60% of their maximal power output (Wmax), with and without an antigravity (anti-G) suit inflated to 52 mm Hg (1 psi). For P, higher preexercise systolic pressure (127 vs 117 mm Hg) was seen with the anti-G suit. At 40 and 60% Wmax, significantly lower heart rates (at 40% = 5.7%; at 60% = 10.6%) at similar cardiac outputs were seen for P with an anti-G suit. Although not significant, P also demonstrated higher stroke volumes at 40% (4.8%) and 60% (5.0%) Wmax with external pressure. For C, no differences in preexercise blood pressure or cardiovascular responses at all three exercise levels were seen with or without the anti-G suit. These data suggest that an inflated anti-G suit is able to prevent venous blood pooling and offers hemodynamic benefits in persons with paraplegia during submaximal arm-cranking exercise. In addition, this study reports a possible alternative to hosiery or functional neuromuscular stimulation that could be applied to all subjects with spinal cord injuries regardless of type or duration of the lesion or of muscle-atrophy.


Aerobic fitness: I. Response of volume regulating hormones to head-down tilt.

We investigated the relationship of aerobic fitness to the response of volume-regulating hormones to acute simulated microgravity. Six untrained (UT) and six endurance-trained (ET) healthy young males were studied in the head-down tilt (HDT) position of -6 degrees for 4 h. Peak oxygen uptake (VO2peak) and plasma volume (PV) were significantly greater in the ET (VO2peak = 61.7 +/- 1.6 ml.min-1.kg-1 and PV = 53.1 +/- 2.8 ml.kg-1) than in the UT (VO2peak = 38.4 +/- 1.7 ml.min-1.kg-1 and PV = 38.8 +/- 1.0 ml.kg-1). Plasma concentrations of atrial natriuretic peptide (ANP), arginine vasopressin (AVP), norepinephrine (NE), renin activity (PRA), and aldosterone (PA) were measured prior to HDT and at minutes 2, 5, 15, 30, 60, 120, 180, and 240 during HDT. PRA and PA significantly decreased during the time of HDT in both groups. The changes in ANP and NE concentrations were not significantly different between the groups nor across time. However, in the ET subjects, the changes in PRA and NE were significantly correlated with the changes in ANP (r = 0.49, P less than 0.01; and r = 0.86, P less than 0.001, respectively); in the UT subjects, the changes in AVP, PRA, and PA were significantly associated with changes in NE (r = 0.34, P less than 0.03; and r = 0.59; and r = 0.53, P less than 0.01, respectively). PV significantly decreased during HDT, and was primarily related to the decrease in PA in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Aerobic fitness: II. Orthostasis and VO2peak following head-down tilt.

To determine whether endurance exercise trained (ET) subjects would experience greater reductions in peak oxygen delivery and orthostatic tolerance (OT) than untrained (UT) subjects, both peak oxygen uptake (VO2peak) during upright bicycle ergometry and tolerance time during 70 degrees head-up tilt (HUT) were compared within and between groups before and after 4 h of -6 degrees head-down tilt (HDT). Eight ET subjects with a mean VO2peak of 61.7 +/- 1.6 ml.kg-1.min-1 were matched for age, height, and weight with eight UT subjects (VO2peak = 38.4 +/- 1.7 ml.kg-1.min-1). Following HDT, decreases in plasma volume (PV) were larger for ET subjects (-3.7 +/- 0.5 ml.kg-1) than for UT subjects (-2.3 +/- 0.3 ml.kg-1), P less than 0.03. Reductions in VO2peak for ET subjects (-5.4 +/- 1.1 ml.kg-1.min-1) were also greater than for UT subjects (-2.4 +/- 0.8 ml.kg-1.min-1), P less than 0.05. The ET (N = 6) subjects also had a significant decrease in OT time (-13.0 +/- 4.2 min) during post-HDT HUT, which was not observed for the UT group (N = 6). A significant inverse correlation was found pre-HDT VO2peak and the change in OT time, r = -0.74, P less than 0.01. The decrease in OT was also significantly correlated to the PV decrease, r = 0.59, P less than 0.04. The UT subjects had significantly augmented pressor responses to HUT manifested by the increases in both HR and MAP following HDT.(ABSTRACT TRUNCATED AT 250 WORDS)


Energy metabolism and fuel utilization.

1) Total energy expenditure during marathon training and competition is among the highest reported for any endurance activity. 2) Although training intensity for the marathon may vary between 60-125% of maximal physical working capacity, elite runners must run consistently at about 80-90% of VO2max in order to achieve successful competitive performances. 3) Earlier studies demonstrated that carbohydrate was the preferential fuel for endurance exercise, but more recent studies have suggested a greater role for lipids than previously thought. Also, proteins may contribute as much as 5-10% of the total energy to a marathon performance. 4) The preponderance of Type I and IIA skeletal muscle fibers among marathon runners and their significantly improved storage capacity for intramuscular lipids and increased mitochondrial density may account for increased reliance on oxidation of fats.


Sports anemia, iron supplements, and blood doping.

1) Athletes tend to have lower hemoglobin concentrations than sedentary counterparts. This has been called sports anemia, a misnomer. 2) Sports anemia is a false anemia and a beneficial adaptation to aerobic exercise, caused by an expanded plasma volume that dilutes red blood cells. 3) Athletes, however, can also develop true anemia, most commonly caused by iron deficiency. True anemia curbs athletic performance, but nonanemic iron deficiency does not. 4) Iron supplements are useful for women endurance athletes who repeatedly develop iron deficiency anemia despite dietary advice. 5) Some endurance athletes today are blood doping by abusing recombinant human erythropoietin (rEPO). They risk dying to win.


Nutrition for the marathon and other endurance sports: environmental stress and dehydration.

1) During exercise, the body's ability to safely regulate internal temperature is influenced by the environment, exercise intensity, clothing, and the athlete's level of fitness and acclimation. 2) Effective thermoregulation during exercise in the heat requires the evaporation of sweat. The onset of sweating during exercise is triggered by an increase in core body temperature. 3) Dehydration compromises cardiovascular and thermoregulatory function, limits physical work capacity, and increases the risk of heat-related health problems. 4) Physiological and behavioral adaptations help the body cope with the combined demands imposed by exercise and environment. 5) Ad libitum fluid intake is insufficient to protect against dehydration.


Benefits of fluid replacement with carbohydrate during exercise.

Ingestion of approximately 30-60 g of carbohydrate during each hour of exercise will generally be sufficient to maintain blood glucose oxidation late in exercise and delay fatigue. Since the average rates of gastric emptying and intestinal absorption exceed 1,250 ml.h-1 for water and solutions containing up to 8% carbohydrate, exercising people can be supplemented with both carbohydrate and fluids at relatively high rates. When cyclists exercise at competitive intensities for 2 h in the heat with a sweat rate of 1,400 ml.h-1, it is clear that the closer that fluid consumption matches sweating rate (at least up to 80% of sweating rate), the better. Increasing dehydration, due to inadequate fluid consumption, directly impairs stroke volume, cardiac output, and skin blood flow, which results in larger increases in body core temperature, heart rate, and ratings of the difficulty of exercise. This same phenomenon probably also applies to running, which argues against the notion that a certain amount of dehydration (i.e., up to 3%) is permissible and without major cardiovascular consequences. However, runners generally drink only 500 ml.h-1 of fluid and thus allow themselves to dehydrate at rates of 500-1,000 ml.h-1. The performance question boils down to "Will the time lost as a result of drinking larger volumes be compensated by the physiological benefits drinking produces and the faster running pace that might be achieved during the last half of the race?" However, if the goal is safety, which means minimizing hyperthermia, there is no question that the closer that the rate of drinking can match the rate of dehydration, the better.


Nutritional manipulations before and during endurance exercise: effects on performance.

1) Ingesting CHO during prolonged, moderate-intensity (60-85% VO2max) exercise can improve performance by maintaining plasma glucose availability and oxidation during the later stages of exercise. 2) Plasma glucose may be oxidized at rates in excess of 1 g.min-1 late in exercise. Athletes therefore need to ingest sufficient quantities of CHO in order to meet this demand. This can be accomplished by ingesting CHO at 40-75 g.h-1 throughout exercise or by ingesting approximately 200 g of CHO late in exercise. Ingesting CHO after fatigue has already occurred, however, is generally ineffective in restoring and maintaining plasma glucose availability, CHO oxidation, and/or exercise tolerance. 3) No apparent differences exist between glucose, sucrose, or maltodextrins in their ability to improve performance. Ingesting fructose during exercise, however, does not improve performance and may cause gastrointestinal distress. 4) The form of CHO (i.e., solid vs liquid) ingested during exercise is unlikely to be important provided that sufficient water is also consumed when ingesting CHO in solid form. 5) Ingesting 50-200 g of CHO 30-60 min before exercise results in transient hypoglycemia early in exercise, but this does not affect the rate of muscle glycogen utilization or, in most people, cause overt symptoms of neuroglucopenia. Whether performance is impaired, unaffected, or enhanced by such pre-exercise CHO feedings remains equivocal. 6) Ingesting 200-350 g of CHO 3-6 h before exercise appears to improve performance, possibly by maximizing muscle and/or liver glycogen stores or by supplying CHO from the small intestine during exercise itself.(ABSTRACT TRUNCATED AT 250 WORDS)


Recovery from endurance exercise.

1) During training for and competition in endurance exercise athletes often significantly reduce both liver and muscle glycogen reserves. 2) Replenishment of muscle glycogen probably occurs preferentially over the replenishment of liver glycogen after exercise. 3) Muscle and liver glycogen can be replenished within 24 h after exercise provided adequate carbohydrate is consumed. 4) To replenish muscle glycogen within 24 h, 8-10 g carbohydrate.kg-1 body weight should be consumed. 5) To optimize muscle glycogen synthesis during the 4-6 h after exercise, carbohydrate must be consumed immediately after exercise and at frequent intervals thereafter. 6) While normal muscle and/or liver glycogen levels can be normalized 24 h after exercise, muscle function may or may not be fully recovered.


Training for endurance sport.

1) An endurance athlete's performance level is determined largely by three physiological variables: maximal aerobic power, lactate threshold, and economy. Training strategies should be directed toward improvement of these factors. 2) Successful endurance training programs typically include an individually constructed combination of three major training strategies. These are long duration, moderate intensity training; moderate duration, high intensity training; and short duration, very high intensity training. 3) Endurance training involves a large daily expenditure of energy, and much of this energy is derived from catabolism of carbohydrates. Accordingly, it is essential that the endurance athlete's diet be rich in complex carbohydrates and provide the total amount of energy needed to maintain energy balance.


Food selection for endurance sports.

1) The body requires at least 40 nutrients that are classified into six groups: protein, carbohydrate, fat, vitamin, mineral, and water. These nutrients cannot be made in the body and so they must be supplied from solid or liquid foods. 2) Fat, carbohydrate, and protein contain energy that is measured in units called kilocalories. Alcohol also contains kilocalories, but is not a recommended energy source for endurance exercise. 3) Foods in endurance sports training programs should provide adequate fluids to prevent dehydration; energy intake that is high in carbohydrate, low in fat, adequate in protein, and that maintains desirable body weight and desirable proportions of fat and lean weight; and sufficient amounts of vitamins and minerals. 4) Six categories of food types form the fundamentals of good diets for endurance exercise training and include: fruits, vegetables, grains-legumes, lean meats, low-fat milk products, and fats-sweets. Vegetarian diets include all food type categories except meat and/or milk products. 5) Fat and carbohydrate content of foods in each food type category varies greatly because of how foods are prepared. 6) The Food Pyramid and Sports Food Swap are guides for selecting foods that provide recommended amounts of essential nutrients for endurance exercise. 7) Before, during, and after endurance exercise, food intake should include adequate amounts of easily digestible, high carbohydrate foods that are familiar and psychologically satisfying. 8) Easily digestible high carbohydrate liquid or solid foods should be eaten soon after exercise is stopped to maximize rates of glycogen replacement. 9) Dehydration can be prevented by adequate fluid intake before, during, and after exercise. 10) Any food plan should be tested before a competition to find out how well that plan works for an athlete.


Prevention and treatment of injuries to runners.

1) Many otherwise healthy runners are prevented from participating fully in their chosen endurance sport because of overuse injuries. 2) The most important risk factor for incurring an overuse injury is a training error, such as excessive mileage, sudden change in training distance or intensity, too much hard interval training, improper footwear, and running on chambered surfaces. 3) Although the knee is the most frequent site of injury in runners, any part of the lower extremity may be affected. 4) Tendinitis, muscle strain, and stress fractures are the most common overuse injuries in endurance athletes. 5) Prevention of injury through elimination of risk factors is clearly preferable to treatment. If injury does occur, treatment should include appropriate medical care, athlete education, cross-training, specific rehabilitative exercises, and a programmed return to running.


[Retrospective study of infection by the human immunodeficiency virus in pregnant women. Future of the child and the mother].

5.4% (108 of 2,000) women have been confirmed for HIV 1 infection in Brazzaville in 1987. 1,172 deliveries have been registered by study, 7.76% have been occurred in HIV+ women. The prevalence of miscarriages was significantly, higher in HIV+ women than in HIV- ones (p less than 0.001). The birth-weight of new-borns was not significantly different among the children born to HIV+ and HIV- mothers. There were the various probable reasons of death of children born to HIV+ mothers. More frequently the respiratory affections with persistent hyperthermia were noticed. During the follow-up, in all groups of age, the fever with failure of thrive were the most frequent signs (50%) with pneumopathy. The clinical picture was completed by diarrhoea after six months of live.


[Endocrinological aspects of acquired immunodeficiency syndrome (AIDS)].

The acquired immunodeficiency syndrome (AIDS) is a severe disease. Lung, brain and digestive complaints that result in opportunistic infections and neoplasms are the most documented. The endocrine disorders, the description of which is recent on the basis of post-mortem data, motivated some authors to undertake studies in order to evaluate endocrine function in patients infected with human immunodeficiency virus (HIV). An increase in serum cortisol level due to stress adaptation in the early stage of the disease is reported, whereas peripheral adrenal insufficiency, although unusual, appears in the late stage. Hypogonadism is common in men with HIV infection but its origin is discussed, central or peripheral. The thyroid function is generally preserved, but low T3 syndrome which correlates with bad prognosis may be observed. Anterior hypopituitarism, which is rare, hyponatremia more frequent because of its etiopathogenic factors in AIDS, constant and precocious hyperprolactinemia, are noticed. The episodes of asymptomatic or severe hypoglycemia may be explained by inanition and side effects of drugs. The non-specificity of the clinical manifestations of endocrine dysfunction masked by the classic signs of AIDS, shouldn't make forget the systematic search of glandular abnormalities as soon as an evocative symptomatology is conspicuous. The mechanisms of endocrine complications are badly known, but opportunistic infections, drugs, particularities related to the structure of HIV and interleukin 1 (IL1) surely play a role. The recognition of endocrine disorders is essential for optimal therapy because, unknown, their evolution is dramatic.


[Congenital malaria].

Several physiopathological hypothesis may explain the frequent transmission of malaria from a pregnant woman to the foetus, and its obstetrical consequences. Because of immunological reasons, such a transmission is most often silent, but some severe forms of congenital malaria do exist and they justify the chemoprophylaxy for pregnant women, and the treatment of any presumptive attack of malaria during pregnancy.


Vascularized fibular graft for reconstruction after resection of aggressive benign and malignant bone tumors.

From 1982 to 1991, 19 patients at Nara Medical University, Kashihara, Japan, underwent resection of aggressive benign and malignant bone tumors, with limb salvage and reconstruction by free vascularized fibula grafts. The patients were followed up for an average of 54 months. The reconstructed site was the jaw in 6 cases, upper extremity in 3, spine in 1, pelvis in 2, and lower extremity in 7. Six patients had aggressive benign lesions, and 13 had malignant lesions. The sizes of the resultant bone defect ranged from 6 to 20 cm, and the lengths of fibular used ranged from 8 to 24 cm. The average time to union was 4.1 months (2-9 months) in the extremities and pelvis. Local recurrence was observed in 3 cases, for whom vascularized fibula grafts were performed for recurrent tumors. In cases of primary untreated tumors, no recurrences occurred. Therefore, this procedure should be performed at the time of primary operation after extensive resection of an aggressive benign or malignant bone tumor.


Vein grafts in reconstructive microsurgery of the lower extremity.

This paper describes surgical techniques and results of lower extremity microsurgical reconstruction using vein grafts. During a 5 year period, free tissue transfers were used to reconstruct 118 lower extremity defects in 109 patients. In 28 of these cases (24%), saphenous vein grafts were used when reconstruction would otherwise have been difficult or impossible. A preliminary arteriovenous fistula was established between the proximal arterial source and the final venous drainage in the majority (26) of cases. Venous drainage was left intact at the point of entry into the deep venous system in 23 cases. Three complications included anastomotic clotting in 2 patients and postoperative blow-out of the vein graft in another; all were treated without flap loss. Flap survival was 86% in vein grafted patients and 94% in those cases in which vein grafts were not used. Flap loss was related in each case to the nature of the pre-existing condition, rather than to vein grafting per se.


[Glomerulopathies in schistosomiasis. Current data].

The authors studied various glomerular injuries in schistosomiasis. They occurred more after with Schistosoma mansoni and rarely with other Schistosoma species. Different lesions were observed: the membrano-proliferative type is frequently noted; but mesangial and extramembranous glomerulitis were also described. The pathology of glomerular lesions is Immune mediated with activation of complement patway and deposit of immune circulating complexes.


[Simultaneous association of tubercular meningitis and cryptococcal meningitis in an African with human immunodeficiency virus HIV positive serology. University Hospital Center of Bujumbura,Burundi].

The authors report a connection between a meningitis tuberculosis and a meningoencephalitis with cryptococcus in the case of an african VIH+. The diagnostic of a meningitis tuberculosis was retained on an indirect arguments, this of meningoencephalitis of direct arguments (antigen cryptococcus, cultivation on Sabouraud environment). The pulmonary tuberculosis and/or extrapulmonary tuberculosis is current in Central Africa during HIV infection, as well as the crytococcosis during AIDS. But, any observation on neuromeningitis strike of those two infections have been reported up to now.


Short- and long-term evaluation of arteriovenous fistulas for chronic hemodialysis.

One hundred ninety-three internal arteriovenous (A-V) fistulas were performed on patients with renal failure in the last eleven years. The A-V fistulas were divided on the basis of the following: side-to-side between the radial artery and cephalic vein (157 cases); end-to-side between the cephalic vein and radial artery (11 cases); end-to-end between the radial artery and cephalic vein (5 cases); A-V fistulas between the brachial artery and the cephalic, basilic or medial cubital vein (20 cases). The side-to-side A-V fistulas achieved an immediate patency in 88% of cases, lower than each of the other groups. However, the subsequent long-term failure rate was less than the other groups. In addition, satisfactory function was maintained at a high level (93%) during the follow-up period. The end-to-side and end-to-end A-V fistulas showed higher percentages of immediate patency (90% and 100%, respectively), although they were used more often on patients with vascular problems. Diabetic nephropathy was the main factor associated with thrombosis of A-V fistulas.


[Preliminary data on dermatomycoses in Ouagadougou (Burkina Faso)].

This study is a contribution to a best knowledge of mycosis in Burkina Faso where the data concerning these diseases are very old. It has been focused on the clinical and mycological features of the dermoskeloton mycosis through the consultations at the two offices of dermatology in the city of Ouagadougou. From April to October, 1990, 216 patients have been taken census of, and they have shown 265 lesions among which 143 mycosis of the skinfolds (54 p. cent), 45 onychomycosis (17 p. cent), 29 palmoplantar mycosis (11 p. cent), 24 mycosis of the glabrous skin (9 p. cent) and 24 mycosis of the scalp (9 p. cent). From these 265 lesions we have isolated 156 strains of dermatophytes and 108 strains of candida. The species which have been seen more frequently are Candida albicans (30 p. cent), Trichophyton rubrum (19 p. cent), Trichophyton soudanense (13 p. cent) and Trichophyton tonsurans (8 p. cent). This study has enabled the identification of some clinical features and some responsible agents of the dermatoskeloton mycosis in Ouagadougou. It will lay the foundation for further specific studies in the country.


Hair growth following scalp microvascular flap transfer for baldness due to burn injury.

A female patient, 8 years of age, presented with baldness of the right scalp following deep scalds from boiling soup landing on the head, neck, and chest. The depth of the burn was severe enough to cause baldness. She was primarily advised to wear a wig to address the problem of baldness on one side. Surgery was planned to use uninjured scalp skin to offer hairy skin coverage of the bald site. A left scalp skin flap (2.5 by 7 cm) based on the superficial temporal artery and vein was transferred to the bald area, with microvascular anastomosis to the superficial temporal vessels on the right side. There was complete survival of the flap with uneventful recovery and satisfactory growth of hair. Hair growth from the flap was comparatively thicker than from the rest of the scalp. This microvascular flap has produced sufficient hair to cover the entire area of the baldness and the patient does not need to wear a wig.


Neck resurfacing with free tram flap.

Anterior neck defects are best reconstructed with flaps, to avoid contractures and extensive scarring. Local or distal flaps are hampered by significant donor site disability. The development of the TRAM flap for breast reconstruction has improved our armamentarium in reconstructive surgery. This paper purports to demonstrate the superiority of the free bilateral TRAM flap applied for anterior neck resurfacing. Two cases with such anterior neck conditions are presented.


[Cardiac manifestations of sickle cell disease].

In this study the authors report the cardiac complications seen in the course of sickle-cell disease. Among 41 homozygot patients the complications reported were as follow. Effort dyspnea in 15/32 (46% cases); systolic souffle in 36 patients (87.8% of cases); cardiomegaly in 30 patients (17% of cases) and electric abnormalities in 70% of cases. The echotomography practised in 27 cases was normal in 12 cases and showed a hyperkinetic aspect in 10 cases, a cardiomyopathy in four cases.


[Peripheral and bone marrow hematological modifications in infection by the human immunodeficiency virus (HIV) in central Africa. Diagnostic and prognostic significance].

From the 24 March 1988 to the 15 June 1989, we noticed the peripheric and medullar haematologic modifications of 46 patients infected by HIV, classified in the group IV of CDC. The haematologic modifications found are: a peripheric cytopenia with a constant anemia often serious, a leuconeutropenia, a mild or missing lymphopenia. In comparison: the bone marrow is often rich in the different lineage with more often a sharp lymphoplasmocytal reaction, sometime very characteristic on the cytological side. 2 myeloid leukemia were diagnosed by participation of the myelogram. At least, the proteinogram showed a major polyclonal hypergammaglobulinemy sometimes monoclonal.


Microsurgical repair of the parotid duct.

Because of its relatively small caliber lumen, traumatic injuries of the parotid duct appropriately lend themselves to microsurgical repair. With the evolution of more modern techniques and materials, inert nylon microsutures placed under magnification permit an ideal atraumatic anastomosis of the divided ends of the parotid duct. Such an exact approach may also obviate the need for long-term stenting across the site of repair, which remains a controversial issue.


Successful replantation of digits and restoration of partial hand function: a case report.

A case of a five-finger amputation and avulsion injury with salvage of two digits combined with island flap coverage of the remaining skin defect is reported. The salvaged digits were replanted onto the ulnar and radial borders of the hand stump and rotated to oppose one another. Recovery of partial hand function is reported.


[Ascariasis of the cervix].

The authors report a case of ascaris lumbricoïdes located in the cervix uterus of a patient suffering from gonococcal salpingitis. Reviewing the literature, they conclude that the most likely way to the parts genitals was a transanal migration toward vagina.


Vascularization of skin following implantation of an arteriovenous pedicle: implications in flap prefabrication.

In a rat model, a skin flap was fabricated by implantation of a distally ligated arteriovenous pedicle. The femoral artery and vein were implanted as a unit beneath the abdominal skin, a portion of which was later raised as an island flap, based on these vessels. Percentage area of survival, tissue blood flow, and pattern of vascularity were compared in two groups of flaps. In group I, the pedicle to be implanted was dissected with a cuff of surrounding muscle still attached; in group II, the pedicle was skeletonized to the level of adventitia. Flap survival in the two groups was similar (73% vs. 77%), as was skin blood flow (5.4 vs. 5.7 ml/100 g/min). Angiography demonstrated two principal patterns of vascularization: connection between donor and recipient vessels (inoculation), and sprouting and arborization of new vessels. Strengths and limitations of this and other models of flap "prefabrication" are discussed.


Further investigation of secondary venous obstruction.

The first ischemic insult a tissue suffers is primary (1 degree). A second ischemic episode, such as thrombosis after free tissue transfer may be regarded as secondary (2 degrees) ischemia. The current study investigated 2 degrees ischemia in rodent epigastric flaps. Flaps were elevated in 50 Sprague-Dawley rats: group 1 had 5 hours 1 degree venous ischemia induced by placement of microvascular clamps; group 2 was like group 1, except venous continuity was re-established by venous anastomosis after resection of the venous segment previously microclamped; group 3 had 15 minutes of 1 degree ischemia, 24 hr later 5 hr of 2 degrees venous ischemia was induced by placement of microvascular clamps; group 4 was like group 3, except the venous segment was excised. Necrosis was evaluated on postoperative day 7. Both secondary ischemic groups had significantly less flap survival than the corresponding primary ischemic groups (P less than 0.001 for both). Resection of a portion of the vein and subsequent microanastomosis did not reduce flap survival (NS). Secondary venous ischemia of 5-hr duration is poorly tolerated by rodent skin flaps. There was no difference in flap survival in those flaps whose veins were clamped for 5 hr compared to those flaps whose clamped venous segments were resected and re-anastomosed.


Carotid blood flow in anesthetized rats: effects of carotid ligation and anastomosis.

An experimental model was developed for continuous measurement of the common carotid blood flow, in the anesthetized rat, with an electromagnetic flowmeter. The mean carotid blood flow for 41 rats averaged 3.2 +/- 0.2 ml/min at an average mean arterial pressure of 101 +/- 2.5 mm Hg and arterial pO2 of 106 +/- 3.4 mm Hg, pCO2 of 38 +/- 1.2 mm Hg, and pH of 7.36 +/- 0.02. This model allowed short- and long-term carotid hemodynamic changes to be recorded after contralateral carotid ligation or end-to-side carotid anastomosis. After ligation of the contralateral carotid, blood flow through the intact carotid increased 25% (at about 1 week) and 45% (at about 5-6 months), whereas, after anastomosis, carotid blood flow increased 76% and 89% at periods comparable to those after ligation. Arterial pressure and gases did not change throughout the experimental periods. Probable mechanisms involved in these carotid hemodynamic changes are discussed.


Vascularized and nonvascularized parathyroid gland transfer: a comparative study.

There is currently no animal model available to study the results of vascularized parathyroid gland autotransfers and nonvascularized autotransfers in the same model. This study was undertaken to determine the technical feasibility of performing microvascular parathyroid gland autotransfers in the dog, and to compare the postoperative levels of calcium, phosphorous, and parathyroid hormone (PTH) levels to a group of dogs who underwent nonvascularized parathyroid gland transfers. Dogs in the nonvascularized group were unable to maintain normal calcium without supplements, whereas dogs in the vascularized group maintained levels in the normal range. Microsurgical techniques allow transplant of a precise amount of parathyroid gland with its vascular supply to another part of the body with almost immediate restoration of function.


Microsurgical use of polydioxanone (PDS) suture: an experimental report.

Although many technological advances have been made in surgical materials, nylon is still the main suture material use for microvascular surgery. This study sought to evaluate polydioxanone (PDS) sutures for use in microvascular anastomoses. Twenty-eight male Sprague-Dawley rats were used in this experiment. Spatula-type needles with 9-0 PDS suture were used to anastomose the right femoral arteries, with 9-0 nylon used on the left side. The arteries were observed for 1, 2, 3, 4, 8, 12, and 16 weeks after surgery to determine arterial patency and to evaluate vascular pathology. Results were comparable between PDS and nylon. We suggest that if the suture material is redesigned to allow smooth passage through the tissue, and if the needle used in conjunction with the suture is improved, PDS may offer an excellent material for microvascular anastomosis.


Chronic cyclosporin A therapy in rats.

We investigated the pharmacokinetics of cyclosporin A (CsA) blood levels and drug toxicity in a chronic rat study in which long-term (30 weeks) CsA was administered. Ninety Lewis rats received subcutaneous CsA at 5 mg/kg/day for 12 weeks, at which time CsA injections were stopped in 50 animals. The remaining 40 rats were maintained on 5 mg/kg CsA daily until week 18 and then switched to an alternate day dosing until week 30. All rats were observed daily and weighed weekly. Whole blood CsA levels were determined by a commercially available radioimmunoassay kit. The daily dosing regimen resulted in greatly elevated trough CsA levels (greater than 1,600 micrograms/liter) and substantial chronic systemic toxicity, with weight loss and death in eight animals. Alternate day dosing reduced trough levels (mean 1,311 micrograms/liter) and decreased toxicity. Chronic administration by the subcutaneous route resulted in a considerable depot effect, with constancy of drug levels over a 48 hr dosing interval and a slow decline of drug levels (15 days) upon cessation of treatment. These results underscore the importance of monitoring both body weight and blood CsA levels in rodent studies when CsA is employed. Investigators should be aware of drug accumulation with chronic therapy and the consequent need to modify dosing to prevent toxicity.


Endothelialization of microporous polytetrafluoroethylene grafts in the infrarenal aorta and caval vein of the rat.

To study healing and endothelialization of vascular grafts, microporous polytetrafluoroethylene (PTFE) prostheses 2 mm in inner diameter and 5 mm long were implanted into the infrarenal aorta or caval vein of the rat. Patency was assessed in six rats from each group at days 3, 7, 14, 28, and 56 after implantation. Four grafts were occluded, two in the aorta (56 days) and two in the caval vein (3 and 14 days). The prostheses were examined via scanning electron and light microscopy for evaluation of endothelialization. At 3 days, the inner surface of the aortic grafts was covered by a plasma proteinaceous layer and that of the caval vein grafts by a thin mural thrombus. Endothelial cells then migrated from aorta/caval vein edges over the graft. At 14 days, the caval vein grafts were completely re-endothelialized, and, at 28 days, the mural thrombus in these grafts was replaced by neointima. In contrast, endothelialization of the aortic grafts had advanced only 1 mm at about 56 days, never forming a complete endothelial layer. We conclude that endothelialization of microporous PTFE prostheses is more rapid and complete in the caval vein than in the aorta of the rat.


Experimental study of free toe replantation in rats.

A rat model for free second toe replantation, free hallux-to-thumb transplantation, and combined free hallux-to-thumb transplantation and free second-toe-to-index transfer is described. Experimental devices, operative methods, and the keys to success are discussed.


Skeletal fixation in the rabbit knee transplantation model.

The immature rabbit knee model for epiphyseal plate transplantations has been used by a number of investigators. Rigid fixation of the femoral osteosynthesis has been difficult to achieve reliably. This paper describes a new technique for obtaining consistent and satisfactory skeletal fixation in this model. The tibia was internally fixed with an ASIF/AO four-hole minifragment plate. The femur was fixed with a 2.4 mm Steinman pin supplemented with two 26 gauge stainless steel interosseous wire loops placed at right angles to each other.


[Toxicological basis for evaluating health risk].

The fundamental premises and toxicological methods used to evaluate exposure to chemicals in the environment have been discussed, with particular regard to the approach and methods for health risk assessment of carcinogens and non-carcinogens. The main disputable problems related to risk assessment and risk estimation have been listed.


[Protein fractions in workers exposed to organic solvents].

Industrial workers exposed to organic solvents were subject to examinations consisting in the assessment of the composition of serum proteins. The analysis revealed frequent shifts in protein fraction pattern. It was noted that the increased concentrations of organic solvents metabolites in urine have been usually accompanied by the increased gamma-globulins levels with a simultaneous decrease in albumin fraction and plasmatic index.


[Does formaldehyde-induced asthma exist?].

Formaldehyde is a ubiquitous airborne pollutant in our modern environment. There are many potential sources of formaldehyde in the industrial setting. Ambient formaldehyde affects primarily the upper airways and eyes. Lower airway and pulmonary effects occur at exposure levels between 6-38 mg/m3. It is so soluble and rapidly metabolized that it seldom reaches the lower respiratory tract to inflict damage. There is no consistent evidence indicating that formaldehyde can be a respiratory sensitizer. It may, on rare occasions, induce bronchial asthma at relatively high exposure doses. There are no conclusive studies that would prove the development of de novo-IgE-mediated respiratory tract symptoms as resulting from inhalation of formaldehyde.


[Local-action vibration and mechanical stress--their hygienic characteristics and medical evaluation of exposed workers].

Medical examination and assessment of the hand-transmitted vibration and mechanical shocks were performed in 69 workers using chipping hammers, grinding tools, pneumatic hammers and forge hammers. Out of the 15 chippers examined, 5 presented vibration-induced white finger syndrome. The latency period for the syndrome was found to be similar to that predicted in the ISO guidelines no. 5349. Twenty seven hammer-using operators complained of joints pain within the upper extremities. The results of the examinations indicate that the work under conditions of mechanical shocks may increase the risk of vibration-induced pathology.


[Principles of medical expert testimony regarding adolescents' capacity for practical vocational training and work].

The presented approach to adolescents' vocational guidance is based on the ILO principle that work should conform to psychophysical conditions of the worker which should be evaluated before the beginning of work. In the paper the main health problems influencing the work capability have been discussed. It is suggested that the student, his parents and tutor should be informed about his health impairments, the degree of disability and therapeutic possibilities. The psychophysical features characteristic of the adolescence justify the health protection against occupational exposure to harmful agents as well as the activities, for health education and health promotion at school.


[Biological testing of the fibrogenic effect of dust from the "Sosnowiec" mine on the lung tissue].

In order to determine biological aggressiveness of settled dusts (mechanical, crumbled) and dusts collected using the gravimetric method, experimental studies were carried out, including: 1) evaluation of the physicochemical parameters (size of dust particles, content of silica, metals and other chemical compounds), 2) evaluation of the haemolytic activity, 3) experimental evaluation of fibrogenic potentials by means of: a) intraperitoneal test--to identity morphological type of reactive changes in the peritoneum and b) intratracheal test--to determine the level of hydroxyproline (collagen) in lungs and the morphological type of reactive changes. Albino rats were used for the experiment. The animals were divided into ten groups which received a single intratracheal injection of 50 mg of mining dust in 0.9% NaCl suspension. Comparative evaluation of biological aggressiveness of mining dusts was conducted basing on the findings of collagen levels in lungs. After the end of the experimental period (3 and 6 months, respectively) histopathological a examination of the lungs and mediastinal lymph nodes was made and the collagen levels in the pulmonary tissue (following Stegemann) were determined. As evidenced by the results of the pathomorphological examination and a statistical analysis: 1) after intratracheal injection the mining dusts induced changes within the respiratory system e.g. inflammatory process and emphysema. The exposure also brought about double increase of collagen level as compared to the control group, 2) histopathological study of the lungs and lymph nodes did not reveal progressive development of fibrogenic changes, 3) cytotoxic test showed differences in the haemolytic activity of settled dust and dusts collected by the gravimetric method.


[Comparative evaluation of the effects of detergents Emulkop and Rokafenol N-8 on the aggressiveness of coal mine dust in the aspect of prevention of its biological effect].

Comparative studies on the impact of the detergents Emulkop and Rokafenol N-8 upon the development and course of experimental pneumoconiosis caused by mine dusts were carried out. Albino rats and rabbits were used for the experiment. Methodological assumptions were based on the analogy with the conditions observed at the workplaces where some means of dust control were used. The acute toxicity evaluation was based on determination of the medial lethal dose (DL50) after intragastric administration of the detergents. Irritating action of the detergents on the skin was also evaluated. The effect of the detergents on the aggressiveness of selected dusts was estimated by means of the intratracheal and intraperitoneal tests. At the end of the experimental period (3 and 6 months, respectively) histopathological investigations of the lungs and mediastinal lymph nodes were carried out, and the hydroxyproline (collagen) levels in the pulmonary tissue were determined. According to Hodge and Sterner Chemical Substance Toxicity Classifications, Emulkop has been classified as non-toxic, and Rokafenol N-8 as a weakly toxic substance. On the basis of the investigation results obtained it has been established that: 1) Rokafenol N-8 does not qualify for use in coal mines as a formulation designed for dust control because it doesn't eliminate coniotic changes and causes a significant increase in the level of hydroxyproline (collagen) in pulmonary tissue, 2) in the case of Emulkop our studies have shown that this detergent can be used to prevent pneumoconiosis in coal mine workers.


[Serpentine mine "Nasławice" as the source of contamination of soil and agricultural plants with metals and fibrous minerals].

The study was aimed at evaluation of the effect of dust, produced at crushing the serpentine, on the soil and crops in Nasławice. The serpentine dust, apart from asbestos, contains also magnesium, calcium, chromium and nickel. These elements have been determined in the soil and plants by means of the standard methods generally used in agricultural chemistry, whereas asbestos content was determined by means of X-ray diffractometry and polarization microscopy. The dust of the crops harvested contained asbestos fibres and, in the plants, the increased content of nickel an chromium was found. The highest amount of asbestos was found in soil in the area at a distance of up to 50 km from the emission source. The soil contains also the increased amounts of chromium and sporadically cadmium.


[Analysis of the incidence of pneumoconiosis among coal miners].

Basing on the prevention system for pneumoconiosis in ++coal mining, an analysis of the disease incidence in the workers of a selected ++coal mine was performed. The subjects were 2.495 miners with the work period longer than 8 years and a group of 822 retired miners. In the former group 16 cases (0.64%) of pneumoconiosis were diagnosed, whereas in the latter as many as 89 cases (10.8%) were found which would indicate a necessity for more detailed examinations in ex-miners of this ++coal mine. The pneumoconiosis changes were detected in the early period of their development. The highest risk was found to refer to longwall and blasting miners and cutter-loadermen. The correlation between radiological changes of the s, t, and u type and the duration of work as well as the high frequency of these changes at highly polluted workplaces seem to point to their relation to dust exposure.


Isolation of phosphorylation-deficient mutants of the Rhizobium meliloti two-component regulatory protein, FixJ.

Rhizobium meliloti FixL and FixJ are members of a symbiotically essential two-component system that regulates nitrogen-fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of the nifA and fixK genes by activating transcription of the nifA and fixK promoters (p-nifA and p-fixK, respectively). In this study, we examined the relationship between the in vivo activity of FixJ as a transcriptional regulator and its ability to be phosphorylated in vitro by the sensor FixL. FixJ mutants were isolated that showed decreased activity on p-nifA in Escherichia coli. Most of the FixJ mutant proteins also showed decreased activity on the fixK promoter. These mutants were analysed in R. meliloti for activity on p-nifA during vegetative growth, where similarities and differences were observed when compared with their phenotypes in E. coli. Three mutants showing significantly less activity in R. meliloti were examined for symbiotic activity in planta and were found to be ineffective. When these three mutant FixJ proteins were examined in vitro for their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings are discussed in relation to signal transduction in the FixLJ system.


Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli.

Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles. The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild-type MBP to these mutant vesicles produced unexpected responses. Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)


Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin.

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.


Cell division in Escherichia coli minB mutants.

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minB mutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re-evaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.


Isolation of arginine auxotrophs, cloning by mutant complementation, and sequence analysis of the argC gene from the cyanobacterium Anabaena species PCC 7120.

Arginine auxotrophs of the dinitrogen-fixing cyanobacterium Anabaena species strain PCC 7120 were isolated after ultraviolet light mutagenesis and penicillin enrichment. Two of these auxotrophs were complemented by a cosmid gene library of the wild-type strain established in Escherichia coli that was transferred en masse to the mutants by conjugation. The gene complementing one of those mutants was found to complement an E. coli argC mutant. Sequencing analysis of the gene showed that it encodes a 322-residue polypeptide that is homologous to the ArgC protein of E. coli, Bacillus subtilis and Streptomyces clavuligerus and to the C-terminal moiety of the Saccharomyces cerevisiae ARG5,6 gene product, N-acetylglutamate semialdehyde dehydrogenase. A cysteine residue present in a highly conserved domain in the five proteins is probably located in the active site of the enzyme. Conserved among the ArgC proteins, sequences resembling the primary structure of nucleotide-binding domains are also found. Downstream of the Anabaena argC gene seven nearly perfect repeats of a heptanucleotide (consensus sequence:5'-CTAATGA-3') are found.


Localization of an immunodominant 64 kDa lipoprotein (LP 64) in the membrane of Mycoplasma gallisepticum and its role in cytadherence.

A 64 kDa lipoprotein (LP 64) haemagglutinin (pI 4.9-5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X-114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]-palmitate-labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post-infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one- or two-dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14C-labelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.


Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr)).

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.


Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy (soyC) and ferredoxinsoy (soyB) from Streptomyces griseus.

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P-450, designated cytochrome P-450soy. A DNA segment carrying the structural gene encoding P-450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P-450soy was compared with that for P-450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe-4S] ferredoxin (soyB), named ferredoxinsoy, was identified.


Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene.

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.


Isolation and characterization of an ndvB locus from Rhizobium fredii.

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).


Chromosomal and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes.

An electrophoretic karyotype has been established for Trichoderma reesei strain QM6a, and several of its derivatives, by pulsed-field gel electrophoresis. All strains examined appear to have seven chromosomes with a total genome size of approximately 33 megabases (Mb). The sizes of the chromosomal bands in strain QM6a are approximately 6.2, 6.0, 5.1, 4.2 (doublet), 3.6 and 3.2 Mb. Genes encoding the cellulase complex and xylanases of T. reesei have been mapped, as have several other genes.


Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene.

Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5'-terminal region of the pac gene demonstrated that both phenylacetic acid induction and catabolite repression by glucose are involved, at the transcriptional level, in the regulation of the pac gene. The studies presented here suggest that this regulation is also present in Escherichia coli transformed strains in which the pac gene was not originally present. Analysis of the nucleotide sequence of the 5'-terminal region of this gene, with a statistical algorithm, confirms that the putative promoter previously proposed by our group is the most feasible within this region. We demonstrate that penicillin acylase activity can confer on E. coli the ability to use penicillin G as a metabolic substrate, by detaching the phenylacetic group which can be used as a carbon source. Based on these data, the regulation properties of the pac gene studied in this work, and the specificity profile of the penicillin acylase enzyme we suggest a role for it in E. coli as a scavenger enzyme for phenylacetylated compounds.


Osmostress-induced changes in yeast gene expression.

When Saccharomyces cerevisiae cells are exposed to high concentration of NaCl, they show reduced viability, methionine uptake and protein biosynthesis. Cells can acquire tolerance against a severe salt shock (up to 1.4 M NaCl) by a previous treatment with 0.7 M NaCl, but not by a previous heat shock. Two-dimensional analysis of [3H]-leucine-labelled proteins from salt-shocked cells (0.7 M NaCl) revealed the elevated rate of synthesis of nine proteins, among which were the heat-shock proteins hsp12 and hsp26. Northern analysis using gene-specific probes confirmed the identity of the latter proteins and, in addition, demonstrated the induction of glycerol-3-phosphate dehydrogenase gene expression. The synthesis of the same set of proteins is induced or enhanced upon exposure of cells to 0.8 M sucrose, although not as dramatically as in an iso-osmolar NaCl concentration (0.7 M).


Decreasing transcription elongation rate in Escherichia coli exposed to amino acid starvation.

The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the 'late' probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth-rate. The transcription elongation rate was c. 43 nucleotides s-1 during exponential growth and decreased abruptly to c. 20 nucleotides s-1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lac-promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA' chains were continued into full-length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s-1 in the unperturbed growth phase to approximately 6 as s-1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.


Disulphide bridge formation in the periplasm of Escherichia coli: beta-lactamase:: human IgG3 hinge fusions as a model system.

We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.


Mutagenesis induced by bacterial UmuDC proteins and their plasmid homologues.

The popular image of a world full of pollutants mutating DNA is only partly true since there are relatively few agents which can subtly and directly change base coding; for example, some alkylating agents alter guanine so that it pairs like adenine. Many more mutagens are less subtle and simply destroy coding altogether rather than changing it. Such mutagens include ultraviolet light, X-rays, DNA cross-linkers and other agents which make DNA breaks or large adducts. In Escherichia coli, mutagenesis by these agents occurs during a DNA repair process which increases cell survival but with an inherent possibility of changing the original sequence. Such mutagenic DNA repair is, in part, encoded by the E. coli umuDC operon. This article reviews the structure, function, regulation and evolution of the umuDC operon and similar genes found both in other species and on naturally occurring plasmids.


(A)BC excinuclease: the Escherichia coli nucleotide excision repair enzyme.

Nucleotide excision repair is the major pathway for removing damage from DNA. (A)BC excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli. In this review, we focus on current understanding of the structure-function of the enzyme and the reaction mechanism of the repair pathway. In addition, recent biochemical studies on preferential repair of actively transcribed genes in E. coli are summarized.


Interrelated effects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter.

The formation of complexes containing high levels of DNA melting at the ribosomal RNA rrnB P1 promoter in vitro is shown to be facilitated by DNA supercoiling or low salt. The effector nucleotide ppGpp is ineffective under these conditions. The loss of supercoils or addition of salt increases the effectiveness of ppGpp in inhibiting formation of these complexes. In vivo plasmid DNA supercoiling is shown to decrease during starvation protocols that also increase levels of ppGpp. The results suggest that ppGpp regulation may be affected by the state of DNA supercoiling in vivo.


Surface protein-CAT reporter fusions demonstrate differential gene expression in the vir regulon of Streptococcus pyogenes.

Streptococcus pyogenes expresses at least two virulence factors, the anti-phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)-derived potential promoter regions of the virR, M protein (emm12), and ScpA (scpA) genes from an M type 12 S. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive trans-acting regulator of emm12 and scpA expression; however, virR is not autoregulated. A potential virR-binding consensus sequence is postulated for emm12, scpA and other M-like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation.


A metalloprotease gene from Streptomyces coelicolor 'Müller' and its transcriptional activator, a member of the LysR family.

A metalloprotease gene (mprA) and its regulatory gene (mprR) from Streptomyces coelicolor 'Müller' DSM3030 were isolated and their DNA sequences determined. The protease secreted by the heterologous host Streptomyces lividans was characterized biochemically as a metalloprotease with a M(r) of 20,000, which is in good agreement with data derived from DNA sequence analysis. The mprA gene is transcribed divergently from mprR, the deduced protein of which displays homology to prokaryotic transcriptional regulators of the LysR family. The regulatory protein (MprR) was shown to bind to the intergenic region between mprR and mprA. It was found to activate transcription of mprA in S. lividans and also in Escherichia coli.


Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis.

The alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis is encoded by four ADH genes. In this paper we report evidence that at least three of these genes are transcribed and translated into protein. KIADH1 and KIADH2, which encode cytoplasmic activities, are preferentially expressed in glucose-grown cells with respect to ethanol-grown cells. KIADH4, which encodes one of the two activities localized within mitochondria, is induced at the transcriptional level in the presence of ethanol as is the ADH2 gene in Saccharomyces cerevisiae. However the regulation of the expression of the K. lactis gene is completely different from that of ADH2 and of other known ADH genes in that KIADH4 is insensitive to glucose repression and is not expressed on non-fermentable carbon sources other than ethanol. This kind of regulation can be clearly observed in non-fermenting strains, where the induction of KIADH4 is dependent on the addition of ethanol to the medium. On the contrary, in fermenting strains KIADH4 is always induced by ethanol or acetaldehyde produced endocellularly and this results in constitutive expression of the gene also in the presence of glucose. The mitochondrial localization of the activity encoded by KIADH4 and the peculiar regulation of this gene could be related to the fact that K. lactis is a petite negative yeast in which some mitochondrial functions seem to be essential for cell viability.


Identification of a novel large extrachromosomal DNA (LED) in the Trypanosomatidae.

We have identified a novel 75 kbp large extrachromosomal DNA (LED) which is stably maintained during developmental conversion of Trypanosoma cruzi. It has a covalently closed circular conformation and is not derived from the kinetoplast network. In all T. cruzi strains analysed, LED contains 18S rRNA and spliced leader (sl) sequences. LED from the T. cruzi Y strain contains a minimum of 15 copies of the sl repeat arrayed in a head-to-tail configuration and 50 copies of a 196 bp repeat. LED is also present in Trypanosoma dionisii (subgenus Schizotrypanosoma) and in other members of the family Trypanosomatidae. LED from different T. cruzi strains and from other members of the Trypanosomatidae differ in their content of large ribosomal subunit rRNA sequences and the 196 bp repeat. The presence of LED in four evolutionarily distant trypanosomatid species suggests that it plays an important role in the biology of these parasites.


The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli.

The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13. The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion. The function of the stable BRP signal peptide was analysed by constructing two plasmids. First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence. Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein. This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing. The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium. Over-expression of the BRP signal peptide was lethal and caused 'lysis'. Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane. These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope.


Polyphosphate-selective porin OprO of Pseudomonas aeruginosa: expression, purification and sequence.

The oprO gene of Pseudomonas aeruginosa codes for a polyphosphate-specific porin and terminates 458 bp upstream of the start codon for the phosphate-specific porin OprP. OprO was found to be expressed only under phosphate-starvation conditions in both wild-type and oprP::Tn501 mutant P. aeruginosa strains. However, unlike the rest of the genes of the Pho regulon, including oprP, expression of oprO required cells to be in the stationary growth phase in addition to phosphate starvation. Wild-type P. aeruginosa cells were grown in fermentor culture under these conditions and fractionated by selective solubilization in octylpolyoxyethylene detergent solution. Solubilized OprO was separated from OprP by application to a Mono Q FPLC column and elution with a salt gradient and shown to be functionally identical to cloned OprO produced in Escherichia coli. DNA sequencing of oprO showed the gene product to be highly homologous to OprP, with 76% identity and 16% conserved substitutions. Most genes of the Pho regulon possess a modified -35 region called the Pho box. Two such elements, separated by 4 bp were found in oprO. DNA sequencing also revealed a second Pho box in the oprP gene with the same spacing.


Expression and mutational analysis of the nucleoid-associated protein H-NS of Salmonella typhimurium.

The H-NS (H1) protein is a major component of bacterial chromatin. Mutations in the hns (osmZ) gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes in an allele-specific manner. H-NS expression was found not to vary with growth phase or growth medium osmolarity. Additionally, 10 independent hns mutations were isolated and characterized. Five of these mutations were the result of an IS10 insertion, each generating a truncated polypeptide. The other five mutations were the same specific deletion of one amino acid, delta Ala46. The various hns mutations exhibited different phenotypes and influenced DNA topology to variable extents. Implications for the mechanism by which H-NS influences gene expression are discussed.


The effects of 5'-capping, 3'-polyadenylation and leader composition upon the translation and stability of mRNA in a cell-free extract derived from the yeast Saccharomyces cerevisiae.

A new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell-free extracts. The system was used to examine the influence of 5'-capping, 3'-polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat-shock protein 26) mRNAs. The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA. The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence. This may indicate the existence of a translational mechanism that enhances cap-independent translation. The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.


Cloning and nucleotide sequence of the csp1 gene encoding PS1, one of the two major secreted proteins of Corynebacterium glutamicum: the deduced N-terminal region of PS1 is similar to the Mycobacterium antigen 85 complex.

Two proteins, PS1 and PS2, were detected in the culture medium of Corynebacterium glutamicum and are the major proteins secreted by this bacterium. No enzymatic activity was identified for either of the two proteins. Immunologically cross-reacting proteins were found in a variety of C. glutamicum strains but not in the coryneform Arthrobacter aureus. The gene encoding PS1, csp1, was cloned in lambda gt11 using polyclonal antibodies raised against PS1 to screen for producing clones. The csp1 gene was expressed in Escherichia coli, presumably from its own promoter, and directed the synthesis of two proteins recognized by anti-PS1 antibodies. The major protein band, of lower M(r), was detected in the periplasmic fraction. It had the same M(r) as the PS1 protein band detected in the supernatant of C. glutamicum cultures and presumably corresponds to the mature form of PS1. The minor protein band appears to be the precursor form of PS1. The nucleotide sequence of the csp1 gene was determined and contained an open reading frame encoding a polypeptide with a calculated molecular weight of 70,874, with a putative signal peptide with a molecular weight of 4411. This is consistent with the M(r) determined for PS1 from C. glutamicum culture supernatant and E. coli whole-cell extracts. The NH2-half of the deduced amino acid is similar (about 33% identical residues and 52% including similar residues) to the secreted antigen 85 protein complex of Mycobacterium. The csp1 gene in C. glutamicum was disrupted without any apparent effect on growth or viability.


Analysis of the regulation of the pelBC genes in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi secretes five major isoenzymes of pectate lyases encoded by the pelABCDE genes. The nucleotide sequence of the region surrounding the pelB gene of E. chrysanthemi 3937 was determined, including the regulatory regions involved in pelB and pelC expression. Analysis of the transcripts showed that transcription of pelB or pelC gave, in both cases, only one transcript. The transcription initiation sites of both pelB and pelC were precisely determined as well as the position of the transcription termination of pelB. The pelB and pelC promoters are very similar, showing a good homology with the -35 consensus region but low homology with the -10 consensus. In both cases a KdgR-box overlaps the -35 region. The pelC gene may have two KdgR operators. Moreover, the pelB and pelC genes are preceded by other sequences presenting the typical symmetry of operator sites that could be involved in more specific regulations. Comparison of E. chyrsanthemi pel regulatory regions revealed three classes of homology: pelA, pelB-pelC and pelD-pelE. The sole regulatory sequence conserved among the three classes corresponds to the KdgR-binding site. Moreover, all the pel regulatory regions are AT-rich in contrast to the coding regions which are GC-rich. Gel retardation experiments with fragments overlapping the pelB or pelC regulatory regions demonstrated that the KdgR protein specifically binds to these regions. Other proteins probably also interact with these DNA fragments. Transcription of pelB terminates in a region corresponding to a GC-rich inverted repeat followed by a run of T residues, typical of rho-independent transcription termination sites. Moreover, preliminary results imply that a region adjacent to pelC provoke, directly or indirectly, the repression of pelB and pelC expression.


The plasmid-encoded chloramphenicol-resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins.

The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.


Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri.

Seven virulence loci have been identified by Tn5 insertion mutagenesis on the large 230 kb plasmid (pMYSH6000) of Shigella flexneri 2a. In this study, we used Tn10 insertion mutagenesis and identified a novel virulence locus on pMYSH6000 responsible for bacterial spread. Characterization of the invading bacteria of the Tn10 insertion mutants in the epithelial cells revealed that the bacteria were capable of at least some intracellular spreading but not intercellular spreading. Immunoblot analysis of lysates of the Tn10 insertion mutants with a VirG-specific antipeptide antibody revealed diminished levels of the 116 kDa VirG protein. The virG mRNA in the mutants, however, was expressed at the same level as that in the wild type. The DNA region required for the virulence phenotype was localized to a 1.6 kb DNA sequence in the SalI-K fragment on the plasmid, and thus the locus was designated virK. Expression of virK in Escherichia coli using a T7 RNA polymerase-dependent promoter system yielded a 36 kDa protein. The nucleotide sequence of 1642 bp encoding VirK function was determined, and an open reading frame encoding 316 amino acid residues was shown to encode the VirK protein. The virK region was highly conserved among the large virulence plasmids of shigellae and enteroinvasive Escherichia coli. These results suggest that VirK function is an essential virulence determinant for shigellae involved in the expression of virG gene product at post-transcriptional level.


Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity.

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.


Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins.

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.


SecY and integral membrane components of the Escherichia coli protein translocation system.

Genetic approaches can address the question of how integral membrane Sec factors interact with each other and facilitate protein translocation across the cytoplasmic membrane of E. coli. This review summarizes genetic analyses of SecY, SecE and some other protein translocation factors, utilizing 'prl' mutations, 'sec' mutations, 'suppressor-directed inactivation', 'Sec titration', dominant negative mutations and their suppressors. Evidence suggests that co-ordinate participation of SecY, SecE, SecD, SecF, and probably some other factors, is crucial for the process.


Stress-induced proteolysis in yeast.

Survival of cells in their natural environment is crucially dependent on their ability to adapt to constantly occurring changes. The ability of cells to respond to extremes of environmental influences is vital to survival. Proteolysis is a central cellular tool in stress response. Proteins of pathways necessary for normal growth, but harmful under stress conditions, as well as proteins damaged by stress have to be eliminated. The yeast Saccharomyces cerevisiae, a model eukaryote, has evolved two different proteolytic systems: (i) a membrane-enveloped, vacuolar (lysosomal) system, which contains a variety of non-specific peptidases and (ii) highly specific peptidases residing at different cellular locations. The best characterized peptidase of the specific system is proteinase yscE, the proteasome equivalent found in all eukaryotic cells. Both the vacuolar and the non-vacuolar systems are vital components of the stress response in yeast.


Isolation and characterization of the aadA aminoglycoside-resistance gene from Salmonella choleraesuis.

The streptomycin- and spectinomycin-resistance gene of Salmonella choleraesuis was cloned and its nucleotide sequence determined. The gene is 789 bases long, encoding a protein of a predicted size of 29,353 Da. The gene product inactivated streptomycin and spectinomycin by an adenylation modification. It is homologous (c. 40% total identity) to streptomycin adenylyltransferase, a 3'(9)-O-nucleotidyltransferase (AAD(3')(9)), which is encoded by the aadA gene in Escherichia coli, Agrobacterium tumefaciens, Klebsiella pneumonia, and Serratia marcescens. The AadA protein of S. choleraesuis differs significantly from the other AadA proteins, indicating that it may have diverged from the other members of this family earlier in evolution. Southern hybridization analysis revealed that homologous aadA sequences were also present in other streptomycin-resistant Salmonella species.


Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae.

Resistance to third-generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin-binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third-generation cephalosporins (greater than 250-fold for cefotaxime; greater than 100-fold for ceftriaxone) in a single step (transformation frequency of about 10(-5)). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.


The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity.

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.


The class 1 outer membrane protein of Neisseria meningitidis produced in Bacillus subtilis can give rise to protective immunity.

The class 1 outer membrane protein of Neisseria meningitidis B:15:P1.7,16 was expressed in Bacillus subtilis in high yield as intracellular aggregates. These were easy to isolate and the protein (called BacP1) could be solubilized under denaturing conditions. Sera of mice immunized with thus-solubilized BacP1 contained high titres of antibodies that reacted with the class 1 protein of the meningococcal envelope in immunoblots but did not react with native meningococcal envelope in enzyme immunoassays (EIA) or with intact meningococci in bactericidal assays. However, when the BacP1 protein was complexed with heterologous (Salmonella) lipopolysaccharide, the ensuing sera reacted with meningococcal envelope preparations in both EIA and immunoblots, showed subtype-specific bactericidal activity, and were protective in an infant rat meningitis model.


Fur regulation in Yersinia species.

The effects of iron have been linked with several phenomena including regulation of membrane proteins; however, the mechanism of iron regulation is not well characterized in Yersinia pestis. It is well known that in Escherichia coli, the fur gene product mediates negative transcriptional regulation of several genes in response to iron. We have cloned a Y. pestis fur gene which is highly homologous to the E. coli fur regulatory gene. The sequence of the Y. pestis fur gene exhibits 75% homology to the E. coli gene at the nucleotide level, and 84% homology at the predicted amino acid level. The Y. pestis fur gene is transcribed as a single gene message of approximately 0.5 kb which encodes an approximately 16 kDa protein when expressed in E. coli minicells. A Yersinia enterocolitica fur mutant exhibits hypersensitivity to the Y. pestis bacteriocin, pesticin; the cloned Y. pestis fur gene restores wild-type levels of pesticin sensitivity. Furthermore, iron regulation of at least five surface proteins in this Y. enterocolitica fur mutant is restored by transcomplementation with the Y. pestis fur gene. These data indicate that Y. pestis and Y. enterocolitica possess homologous Fur systems which regulate expression of proteins in response to iron availability.


Quantitative determination of FtsA at different growth rates in Escherichia coli using monoclonal antibodies.

FtsA is an essential cell division protein in Escherichia coli. Its synthesis in low amounts makes the investigation of its functions difficult. Partially purified FtsA protein was obtained by solubilizing cellular inclusion bodies after overexpression of the ftsA gene for the purpose of raising monoclonal antibodies. Mice were immunized with this FtsA protein fraction and their spleen cells were fused to Sp2/0-AG14 mouse myeloma cells. Hybrid cells were screened and two clones were positively identified as FtsA monoclonal antibody producers by enzyme-linked immunosorbent assay and Western blotting. A quantitative assay using these monoclonal antibodies indicated that the average number of FtsA molecules per cell to be between 50 and 200. However, the concentration of FtsA protein normalized to total cell protein was constant over a wide range of growth rates. This finding is in agreement with the hypothesized role of FtsA protein as a stoichiometric component of the septum.