Phosvitin kinase activity in Acholeplasma axanthum.

Incubating the soluble fraction derived from A. axanthum with phosvitin and [gamma-32P]ATP results in the phosphorylation of phosvitin. Casein, histone and kemptide were practically ineffective substrates, whereas a 55 kDa protein of M. gallisepticum was efficiently phosphorylated. The enzymatic activity has an optimal pH in the pH range 6.0-6.2 and requires divalent cations. The activity was inhibited by ammonium sulfate, heparin and sulphydryl blocking reagents, but was not affected by calmodulin with or without Ca2+ or by cyclic AMP.

Increased recovery of group B Streptococcus by the inclusion of rectal culturing and enrichment.

Detection of intrapartum carriage of group B streptococcus (GBS) and subsequent antibiotic prophylaxis may prevent GBS infections in neonates. Because the gastrointestinal tract is the primary source of this organism, detection of carrier status requires both rectal and vaginal swabs. Vaginal swabs from 651 obstetric outpatients were plated onto 5% sheep blood agar. A second vaginal and a rectal swab were collected and incubated overnight in an enrichment medium of Todd-Hewitt broth containing antibiotics. By at least one method, 110 (16.9%) patients were positive for GBS. Only 31.8% of these positive patients were detected by direct culture of vaginal swabs. The use of vaginal swabs directly plated onto blood agar identified only three carriers not detected by another method. Inoculation of an enrichment broth with the vaginal swab and subsequent subculture detected 70.9% of the total. The use of both vaginal and rectal swabs with enrichment detected 97.3% of total GBS carriers. A subset of enrichment broths inoculated with vaginal and rectal specimens from 279 patients was tested for GBS by direct latex agglutination (Streptex; Murex Diagnostics, Inc., Norcross, GA, USA). Of the 90 broths that grew GBS on subculture, only 59 (65.6%) were positive by the direct agglutination method. The use of this method, although reducing processing time by 1 day, gave false-negative results for one-third of the GBS-positive broths. An accurate detection of the GBS carrier state can only be achieved by a combination of vaginal and rectal swabs incubated in enrichment broth and subcultured on blood agar.

The effects of growth in human serum on an acapsular group B Streptococcus mutant.

A Group B Streptococcus Type III (GBS) mutant which, when grown in Todd Hewitt broth (THB), does not produce any detectable capsule, produced a clearly visible polysaccharide capsule when grown in human serum. We isolated cytoplasmic membranes from GBS and separated the component membrane proteins by polyacrylamide gel electrophoresis. A significant change in membrane composition was found during growth in human serum. Several unique proteins were produced on serum growth and there was both up- and down-regulation of other proteins. We measured the intracellular levels of sialic acid for a variety of GBS serotype III isolates. Interestingly, while there was little difference between the intracellular sialic levels of most isolates, the sialic acid level of COH31-15 grown in THB was over 100% higher than that of any other isolate. When grown in serum this pool was reduced to a level similar to that in other strains. The concentration of bacterial cell sialic acid was directly correlated with the sialic acid content of the serum. Exogenous sialic acid content, in concert with other serum factors, plays a role in determining the capsular size in GBS.

The UK accelerated immunisation programme and sudden unexpected death in infancy: case-control study.

OBJECTIVES: To investigate whether the accelerated immunisation programme in the United Kingdom is associated, after adjustment for potential confounding, with the sudden infant death syndrome. DESIGN: Population based case-control study, February 1993 to March 1996. Parental interviews were conducted for each death and for four controls matched for age, locality, and time of sleep. Immunisation status was taken from records held by the parents. SETTING: Five regions in England with a combined population of over 17 million. SUBJECTS: Immunisation details were available for 93% (303/325) of infants whose deaths were attributed to the sudden infant death syndrome (SIDS); 90% (65/72) of infants with explained sudden deaths; and 95% (1515/1588) of controls. RESULTS: After all potential confounding factors were controlled for, immunisation uptake was strongly associated with a lower risk of SIDS (odds ratio 0.45 (95% confidence interval 0.24 to 0.85)). This difference became non-significant (0.67 (0.31 to 1.43)) after further adjustment for other factors specific to the infant's sleeping environment. Similar proportions of SIDS deaths and reference sleeps (corresponding to the time of day during which the index baby had died) among the controls occurred within 48 hours of the last vaccination (5% (7/149) v 5% (41/822)) and within two weeks (21% (31/149) v 27% (224/822)). No longer term temporal association with immunisation was found (P=0.78). Of the SIDS infants who died within two weeks of vaccination, 16% (5/31) had signs and symptoms of illness that suggested that medical contact was required, compared with 26% (16/61) of the non-immunised SIDS infants of similar age. The findings for the infants who died suddenly and unexpectedly but of explained causes mirrored those for SIDS infants. CONCLUSIONS: Immunisation does not lead to sudden unexpected death in infancy, and the direction of the relation is towards protection rather than risk.

Babies sleeping with parents: case-control study of factors influencing the risk of the sudden infant death syndrome. CESDI SUDI research group.

OBJECTIVE: To investigate the risks of the sudden infant death syndrome and factors that may contribute to unsafe sleeping environments. DESIGN: Three year, population based case-control study. Parental interviews were conducted for each sudden infant death and for four controls matched for age, locality, and time of sleep. SETTING: Five regions in England with a total population of over 17 million people. SUBJECTS: 325 babies who died and 1300 control infants. RESULTS: In the multivariate analysis infants who shared their parents' bed and were then put back in their own cot had no increased risk (odds ratio 0.67; 95% confidence interval 0.22 to 2.00). There was an increased risk for infants who shared the bed for the whole sleep or were taken to and found in the parental bed (9.78; 4.02 to 23.83), infants who slept in a separate room from their parents (10.49; 4.26 to 25.81), and infants who shared a sofa (48.99; 5.04 to 475.60). The risk associated with being found in the parental bed was not significant for older infants (>14 weeks) or for infants of parents who did not smoke and became non-significant after adjustment for recent maternal alcohol consumption (>2 units), use of duvets (>4 togs), parental tiredness (infant slept 2 people per room of the house). CONCLUSIONS: There are certain circumstances when bed sharing should be avoided, particularly for infants under four months old. Parents sleeping on a sofa with infants should always be avoided. There is no evidence that bed sharing is hazardous for infants of parents who do not smoke.

In vivo hemolytic activity of group B streptococcus is dependent on erythrocyte-bacteria contact and independent of a carrier molecule.

Experiments were performed to determine the interaction between the hemolysin of group B streptococcus (GBS) and sheep erythrocytes. Growing GBS were shown to possess a potent hemolysin at a very early stage of the growth cycle. After separation of the cells from the growth medium, all the hemolytic activity remained with the bacterial cells, and no activity could be detected in the growth medium. When fetal calf serum was added to the media, some "soluble" activity was detected. This activity, however was completely removed by ultracentrifugation, the hemolytic activity being found solely in the pellet. After the hemolysin had formed, no new protein synthesis was needed to cause hemolysis because the addition of chloramphenicol to cells caused no difference in their hemolytic potential. For proof that no short-lived, soluble factors are produced by the bacteria, bacteria and sheep erythrocytes were incubated in contiguous media, separated by a 0.22-microns membrane. No hemolytic activity was detected on the erythrocyte side of the membrane, although high amounts of hemolysin could be extracted from the bacteria. Only when a detergent was added to the growth medium was hemolysis detected from the erythrocytes, showing that extracted hemolysin could indeed pass through the membrane. These results suggest that the hemolysin is attached to the surface of the cell and that contact is needed between the bacteria and erythrocyte to cause lysis. Where soluble activity was detected, it was connected to bacterial fragments.

Mivacurium in short to intermediate surgical procedures.

Mivacurium, a new benzylisoquinoline muscle relaxant, appears to be close to the ideal for short to intermediate surgical procedures. Ideal properties of such an agent are discussed, in addition to indications for muscle relaxation in such procedures. Two studies are presented, showing the onset and offset times of mivacurium and its cardiovascular stability in both young and elderly patients. It is concluded that it is a well-tolerated and appropriate agent for use in short to intermediate surgical procedures in those patients with normal plasma cholinesterase function, despite a slight prolongation of action in the elderly.

Group B streptococcal disease in the perinatal period.

Group B streptococcus is the major cause of neonatal sepsis in the United States. It is estimated that 2,000 infants die annually of syndromes related to group B streptococcus infection. In the early-onset syndrome, transmission is from mother to child, either in utero or during birth. Pneumonia is the most common presentation in infants who develop symptoms during the first seven days of life. The principal manifestation of late-onset infection is meningitis, which occurs in 85 percent of this group. Although group B streptococcus infection is normally remediable with penicillin therapy, rapid diagnosis and treatment are necessary to prevent the serious consequences of the disease. A vaccine is under development, although the cost-effectiveness of a widespread immunization program for a disease with such a low frequency is still unknown. The potentially serious outcomes of this infection, however, make it a major problem for physicians involved in neonatal care.

Antifungal effects of Allium sativum (garlic) extract against the Aspergillus species involved in otomycosis.

Otomycosis due to saprophytic keratolytic fungi represents a small percentage of clinical external otitis. Although there are certain antibacterial and antifungal agents available, they usually are very caustic, potentially ototoxic and cannot be used if the ear drum is perforated. Garlic is utilized as a folk medicine in many countries for its antimicrobial and other beneficial properties. In response to a lack of otic preparations, the authors studied the efficacy of garlic extracts against the fungi belonging to the genus Aspergillus which are the most common cause of this infection. Aqueous garlic extract (AGE) and concentrated garlic oil (CGO) along with various commercial garlic supplements and pharmaceutical prescriptions were used in an in-vitro study. AGE and especially CGO were found to have antifungal activity. These agents showed similar or better inhibitory effects than the pharmaceutical preparations and demonstrated similar minimum inhibitory concentrations.

Neonatal hypoglycaemia: changing attitudes in the Northern Region.

There has been a recent resurgence of interest in the significance of moderate hypoglycaemia in neonates; although old assumptions have been questioned, there are insufficient data for new and authoritative guidelines to be published. This longitudinal study was designed to test the hypothesis that there have been changes in attitudes among those caring for newborn babies in the 18 maternity units in one health Region. The data were collected by questionnaire in 1986 and 1991, and responses were complete. The results showed a significant shift to higher values in the definition of hypoglycaemia over the five years, in association with a tendency to feed babies earlier--although some units had not altered their policies.

Reaction of 4-methylumbelliferylguanidinobenzoate with cultivated human skin fibroblasts derived from patients with cystic fibrosis.

Protease activity in cultivated human skin fibroblasts has been quantitated using 4-methylumbelliferylguanidinobenzoate (MUGB), an active site titrant of trypsin-like proteases (7). The reaction of the proteases with MUGB was complete in 1 hr, inhibited both by benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate, but not by p-hydroxymercuribenzoate. The extent of reaction was proportional to protein concentration and independent of MUGB concentration. This activity was present in the particulate fraction of the cell. The mean "titre" values (nanomoles of 4-methylumbelliferone released per mg protein) of the proteases in fibroblasts from eight controls (N), 8 obligate heterozygotes (H), and 14 patients with cystic fibrosis (CF) were: N, 1.27 +/- 0.11; H, 0.82 +/- 0.12; CF, 0.66 +/- 0.10. The differences in the "titre" values for N:CF and N:H were significant (p less than 0.001) as were those for H:CF (p less than 0.01). The mean "titre" value obtained for cultivated control amniotic fluid cells was 1.29 +/- 0.17. These data indicate a reduction in the MUGB-reactive proteases in skin fibroblasts derived from patients with CF when compared either to control or to obligate heterozygotes. These data are consistent with our earlier suggestion (11, 15) that decreased proteolytic levels in the tissues and fluids of patients with CF may be a generalized phenomenon.

A 57-kilodalton protein associated with Spiroplasma melliferum fibrils undergoes reversible phosphorylation.

Phosphorylation of a major 57-kilodalton protein substrate was observed in cell lysates of Spiroplasma melliferum BC3 incubated with [gamma-32P]ATP. Only serine phosphates have been isolated from the acid hydrolysate of the phosphorylated protein. The 57-kilodalton protein substrate was found, to a large extent, in the cytosolic fraction and, to a lesser extent, associated with cell membranes and was detected in the Triton X-100-insoluble fraction that contained fibrils.

Growth of group B streptococci in human serum leads to increased cell surface sialic acid and decreased activation of the alternative complement pathway.

Group B streptococcus type III is a major cause of neonatal death. The terminal sialic acid moiety of the group B streptococcus type specific capsule has been shown to be an important virulence factor. We demonstrate here that bacteria grown in human serum have increased cell surface sialic acid content compared with cells grown in common laboratory media. This sialic acid was removed by incubation with neuraminidase, showing that it was on the bacterial surface. Serum-dependent sialylation was dependent on metabolic activity, as the addition of chloramphenicol reduced the amount of added sialic acid by more than 90%. Probing the cell surface with an antibody specific for group B streptococcus type III capsular sialic acid showed an increase in antibody binding after growth in human serum. This effect could be lowered by incubating serum-grown cells in neuraminidase prior to antibody exposure. A group B streptococcus mutant that when grown in laboratory media lacks cell surface sialic acid showed significant cell surface sialic acid when grown in human serum. This increase was associated with a significantly decreased ability to bind C3 and hence activate the alternative complement pathway.

Surgery for a fractured femur and elective ICU admission at 113 yr of age.

Disability and medical dependence increase with age and as the percentage of the population over 80 yr old increases, demands on our healthcare budget will likewise grow. Clinical decision making should always balance the physiological state of the patient against the likely prognosis of the pathological state. Age may influence this decision-making process in an indirect way, as a perceived measure of physiological status, but whether age alone may be used as criteria for the type or degree of clinical intervention is a contentious point. In light of these issues, we present a woman of 113 yr, admitted to hospital with a fractured shaft of femur. The patient underwent surgery and was electively admitted to the intensive care unit for postoperative treatment. She survived surgery and several complications and was subsequently returned to the community where she celebrated her 114th birthday.

Use of student-centred, computer-mediated communication to enhance the medical school curriculum.

OBJECTIVES: While it is clear that computers will play an important role in the study and practice of medicine their introduction into the curriculum remains controversial. Computer purchase has been made compulsory for incoming students. DESIGN: Members of the incoming class were allowed to purchase any computer and modem capable of using the communication program chosen by the school. No formal computer training was given. Students were encouraged to call for assistance or bring in their computers for configuration. The primary object of the system was for communication between the students and between students and faculty. SETTING: The School of Medicine of the University of New Mexico. SUBJECTS: First-year medical students. RESULTS: The vast majority of students set up their computers and connected to the system with little assistance. At the end of the first week of studies all the students were connected. Most of the students used the system on a daily basis. The greatest interest was in discussions concerning examinations with 93% of students reading these postings. The least-used aspect of the system was the exchange of learning issues from small group case discussions. Students also downloaded the curricular material provided but were discriminating in accessing this content. CONCLUSIONS: The student use of the computer as a communication tool has been a success. Students used the system in a variety of ways and by so doing also learned the basics of computer use and maintenance. The area of faculty training is often ignored but is considered crucial to the success of such a project.

Reaction of 4-methylumbelliferylguanidinobenzoate with proteases in plasma of patients with cystic fibrosis.

Protease activity, assayed using 4-methylumbelliferylguanidinobenzoate, an active site titrant of certain proteases, is significantly deficient in plasma of patients with cystic fibrosis. The deficiency can be demonstrated with both chloroform-ellagic acid activated plasma in which the proteases can hydrolyze esters of arginine and unactivated plasma in which the proteases have negligible activity towards these esters. The deficiency can also be demonstrated by separation of the proteases by isoelectric focusing on polyacrylamide gels or by chromatography on agarose columsn. Since protease deficiency can be demonstrated with unactivated plasma, the deficiency in cystic fibrosis is probably due to a reduced number of protease molecules rather than their decreased catalytic efficiency.

Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti.

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.

Protein phosphorylation in Mycoplasma gallisepticum.

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile.

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.