Biomedical subjects

M D Baker

Publications and source records attributed to M D Baker.

At least 37 records · Page 2Linked to original sources

PubMed2001

The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events.

In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu and Cdelta flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cmu and Cdelta regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu and Cdelta regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.

Animals↗

PubMed2001

Effects of Medicaid managed care on health care use: infant emergency department and ambulatory services.

OBJECTIVE: Many urban children rely on emergency departments (ED) for ambulatory care. The objective of this study was to determine whether enrollment in Medicaid managed care (MMC) alters ED or other ambulatory care compared with fee-for-service Medicaid (FFSM). METHODS: A prospective cohort study of infants born between May 1994 and April 1995 with a 6-month follow-up period was conducted in an urban, teaching hospital and surrounding ambulatory settings. A consecutive sample of 644 infants enrolled in MMC or FFSM was studied; 92% of eligible patients were enrolled, and 94% completed follow-up. The main outcome measures were 1) proportion of patients in each group visiting an ED, primary care practitioner (PCP), or specialist; 2) mean number of visits per group; and 3) ED reliance (EDR) defined as the proportion of all ambulatory visits occurring in an ED. RESULTS: Fifty-six percent of MMC and 54% of FFSM patients visited an ED (relative risk: 1.03; 95% confidence interval [CI]: 0.83, 1.27). More MMC patients had a sick visit to their PCP (relative risk: 1.34; 95% CI: 1.03, 1.74); no difference in proportion with well-child or specialty visits was found. Although the mean number of total ambulatory, ED, and specialty visits was the same, MMC patients had fewer well-child and more sick visits to the PCP than FFSM patients (P =.01). EDR was 21% for both groups (P =.95). After adjustment for other factors in multivariate analysis, insurance status remained unassociated with EDR (adjusted odds ratio [OR]: 0.91; 95% CI: 0.56, 1.69). Factors associated with EDR included United States-born mother (OR: 5.34; 95% CI: 1.61, 17.68) and use of a hospital-based primary care physician (OR: 2.00; 95% CI: 1.34, 2.98). Variables that characterized infants who were less likely to be ED reliant included adequate maternal prenatal care (OR: 0.52; 95% CI: 0.34, 0.78) and having a mother who completed high school (OR: 0.67; 95% CI: 0.45, 0.99). CONCLUSIONS: Enrollment in MMC did not alter ED usage patterns when compared with FFSM. Some variation in use of other ambulatory services was detected.

Ambulatory Care↗

PubMed2000

Charged-particle multiplicity near midrapidity in central Au+Au collisions at sqrt[SNN]=56 and 130 GeV.

We present the first measurement of pseudorapidity densities of primary charged particles near midrapidity in Au+Au collisions at sqrt[s(NN)] = 56 and 130 GeV. For the most central collisions, we find the charged-particle pseudorapidity density to be dN/deta|(|eta|<1) = 408+/-12(stat)+/-30(syst) at 56 GeV and 555+/-12(stat)+/-35(syst) at 130 GeV, values that are higher than any previously observed in nuclear collisions. Compared to proton-antiproton collisions, our data show an increase in the pseudorapidity density per participant by more than 40% at the higher energy.

Journal Article↗

PubMed2000

Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting.

In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.

Base Sequence↗

PubMed2000

Axonal flip-flops and oscillators.

The strange and unpleasant sensations (paraesthesiae) or asynchronous motor-unit activation (fasciculation) that result from a period of limb ischaemia are examples of ectopic discharge in peripheral nerves. Ectopic activity also results from demyelination and is associated with serious neurological conditions, such as multiple sclerosis. A build-up of extracellular K(+) in the internode and persistent Na(+) currents are now implicated in generating the different forms of activity arising in normal and demyelinated axons.

Animals↗

PubMed2000

Selective block of late Na(+) current by local anaesthetics in rat large sensory neurones.

The actions of lignocaine and benzocaine on transient and late Na(+) current generated by large diameter (> or =50 microm) adult rat dorsal root ganglion neurones, were studied using patch-clamp techniques. Both drugs blocked whole-cell late Na(+) current in a concentration-dependent manner. At 200 ms following the onset of a clamp step from -110 to -40 mV, the apparent K for block of late Na(+) current by lignocaine was 57.8+/-15 microM (mean+/-s.e.mean, n = 4). The value for benzocaine was 24.9+/-3.3 microM, (mean+/-s.e. mean, n = 3). The effect of lignocaine on transient current, in randomly selected neurones, appeared variable (n = 8, half-block from approximately 50 to 400 microM). Half-block by benzocaine was not attained, but both whole-cell (n = 11) and patch data suggested a high apparent K,>250 microM. Transient current always remained after late current was blocked. The voltage-dependence of residual late current steady-state inactivation was not shifted by 20 microM benzocaine (n = 3), whereas 200 microM benzocaine shifted the voltage-dependence of transient current steady-state inactivation by -18.7+/-5.9 mV (mean+/-s.e.mean, n = 4). In current-clamp, benzocaine (250 microM) could block subthreshold, voltage-dependent inward current, increasing the threshold for eliciting action potentials, without preventing their generation (n = 2). Block of late Na(+) current by systemic local anaesthetic may play a part in preventing ectopic impulse generation in sensory neurones.

Anesthetics, Local↗

PubMed2000

Use of a small palindrome genetic marker to investigate mechanisms of double-strand-break repair in mammalian cells.

We examined mechanisms of mammalian homologous recombination using a gene targeting assay in which the vector-borne region of homology to the chromosome bore small palindrome insertions that frequently escape mismatch repair when encompassed within heteroduplex DNA (hDNA). Our assay permitted the product(s) of each independent recombination event to be recovered for molecular analysis. The results revealed the following: (i) vector-borne double-strand break (DSB) processing usually did not yield a large double-strand gap (DSG); (ii) in 43% of the recombinants, the results were consistent with crossover at or near the DSB; and (iii) in the remaining recombinants, hDNA was an intermediate. The sectored (mixed) genotypes observed in 38% of the recombinants provided direct evidence for involvement of hDNA, while indirect evidence was obtained from the patterns of mismatch repair (MMR). Individual hDNA tracts were either long or short and asymmetric or symmetric on the one side of the DSB examined. Clonal analysis of the sectored recombinants revealed how vector-borne and chromosomal markers were linked in each strand of individual hDNA intermediates. As expected, vector-borne and chromosomal markers usually resided on opposite strands. However, in one recombinant, they were linked on the same strand. The results are discussed with particular reference to the double-strand-break repair (DSBR) model of recombination.

Animals↗

PubMed2000

Mechanisms involved in targeted gene replacement in mammalian cells.

The "ends-out" or omega (Omega)-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal mu-delta locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.

Animals↗

PubMed2000

Use of out-of-hospital interventions for the pediatric patient in an urban emergency medical services system.

OBJECTIVE: To determine appropriateness of out-of-hospital interventions by emergency medical services (EMS) personnel on children with respiratory illnesses. METHODS: A retrospective, cross-sectional study was performed on a random sample of 304 children transported by an urban EMS system during 1994. Data were abstracted from EMScan (a computerized database of all EMS dispatches) and the EMS narrative records. Appropriate utilization of interventions was determined by comparison with the standard EMS protocol for respiratory complaints in this system. An assessment of whether interventions were inappropriately underutilized or inappropriately overutilized was made. Effect of severity of illness, patient age, transport times, and use of medical command on the use of interventions was evaluated. RESULTS: Two hundred three patients (67%) were classified as having respiratory distress. Overall, 56% of the patients received appropriate interventions, 39% received one or two inappropriate interventions, and 5% received three or more inappropriate interventions. Rates of inappropriate utilization with 95% CI for each intervention were: oxygen 16% (95% CI = 12 to 20), assisted ventilation 2% (95% CI = 0.5 to 4), medication use 9% (95% CI = 6 to 13), vascular access 11% (95% CI = 7 to 14), phlebotomy 9% (95% CI = 6 to 13), and cardiac monitoring 18% (95% CI = 14 to 22). Oxygen and medications were underutilized (p < 0.005), whereas vascular access, cardiac monitoring, and phlebotomy were overutilized (p < 0.005). Online medical command (used in 9% of transports) improved appropriate use of vascular access [OR 8.3 (95% CI = 3 to 25) (p < 0.001)] and cardiac monitoring [OR = 3 (95% CI = 1 to 8) (p < 0.05)]. CONCLUSIONS: Emergency medical services personnel underutilized oxygen and medications and overutilized vascular access, phlebotomy, and cardiac monitoring in children with respiratory illness in this urban setting. Increasing patient age, transport times, and illness severity tend to increase the use of certain interventions, while contact with online medical direction seems to improve appropriate use of interventions.

Adolescent↗

PubMed1999

Unpredictability of serious bacterial illness in febrile infants from birth to 1 month of age.

BACKGROUND: A prior study (N Engl J Med. 1993; 329: 1437-1441) produced an effective screen to identify 1-to 2-month-old febrile infants (FIs) who are at low risk of having a serious bacterial illness (SBI). Because of anticipated differences in the epidemiological features of febrile illnesses, that Philadelphia protocol was not applied to FIs younger than 1 month. OBJECTIVES: To describe the epidemiological features of febrile illness in neonates from birth to 1 month of age and to determine the applicability to this population of the Philadelphia screen for identifying FIs at low risk for SBI. DESIGN: A 36-month consecutive cohort study. SETTING: An urban pediatric emergency department. PARTICIPANTS: Infants aged from 3 to 28 days old with temperatures of 38 degrees C or higher. INTERVENTIONS: Following full evaluation for SBI, all FIs, pending results of bacterial cultures, were admitted to the hospital and empirically administered antibiotics. After their illnesses resolved, the medical records of all FIs were reviewed. At that time, the Philadelphia protocol (originally developed for 1- to 2-month-old FIs) was applied and retrospectively judged for safety and efficacy. RESULTS: Of the 254 FIs enrolled, 32 (12.6%) had an SBI. The spectrum of bacterial and nonbacterial diseases closely approximated that described in 1- to 2-month-old FIs. When the Philadelphia protocol was applied to all 254 FIs, 109 (42.9%) would have been identified as at low risk for bacterial disease. Included in that group are 2 FIs with bacterial urinary tract infection, 2 FIs with bacteremia, and 1 FI with bacterial gastroenteritis. CONCLUSIONS: The cause of febrile illnesses in neonates (infants younger than 1 month) approximates that of FIs 1 to 2 months of age. Unlike that for older 1- to 2-month-old FIs, however, the Philadelphia protocol lacks the sensitivity and negative predictive value to identify neonates at low risk for SBI.

Bacterial Infections↗

PubMed1999

Evaluation and management of infants with fever.

Fever is an important symptom in children. In infants younger than 2 months of age, fever might be the only indicator of bacterial disease. Critical to the well-being of these infants is that the physician understand the importance of the occurrence of fever within the first 2 months of life and appropriately investigate the causes of fever. Although all infants with fever require comprehensive evaluation for bacterial disease, not all require hospitalization or empiric treatment with antibiotics. Febrile infants 1 to 2 months of age who, after comprehensive clinical and laboratory evaluation, are found to have no other findings that place them in a higher-risk category for bacterial disease are candidates for continuing observation as outpatients. This management option should be restricted to selected infants whose caretakers and primary physicians agree with and are able to carry out such a plan.

Fever↗

PubMed1999

Utility of portable chest radiographs as a predictor of endotracheal tube cuff pressure.

Increased endotracheal tube cuff pressure causes mucosal ischemia that can lead to necrosis, infection, and, eventually, tracheomalacia or tracheal stenosis. Endotracheally intubated patients frequently undergo portable chest radiography. In this study we explored the relationship of endotracheal tube cuff pressure and the appearance on the tracheal air columns on the portable chest radiograph. We measured the endotracheal tube cuff pressure of intensive care unit patients 124 times immediately before portable chest radiography. On 64 of these radiographs we measured the width of the tracheal air column below the tip of the endotracheal tube and at the maximal diameter of the endotracheal tube balloon. We then analyzed the relationship of cuff pressure to tracheal dilation. The results of ANOVA of tracheal dilation for three groups (safe, borderline, and unsafe cuff pressures) were significant. Large overlapping ranges existed in each group. Regression analysis confirmed a linear relationship between cuff pressure and tracheal dilation (r = 0.435, p < 0.001). Predicted tracheal expansion at 20 mm Hg was a poor screen for endotracheal tube cuff inflation safety; the sensitivity was only 56% and specificity only 71%. The differences in the capacity for tracheal distension between patients make these findings not unexpected. The portable chest radiograph is a poor screening tool for unsafe endotracheal tube cuff pressure.

Adult↗

PubMed1999

The pH dependence of late sodium current in large sensory neurons.

The effects of altering extracellular pH on late Na+ currents were investigated in large dorsal root ganglion neurons from rats (100-300 g), using patch-clamp techniques. The late current amplitude was steeply dependent upon pH over a range which included normal physiological values: raising the pH from 7.3 to 8.3 approximately doubled the amplitude. Whole-cell late currents 60 ms after depolarization to - 30 mV were blocked with an apparent pKa of 6.96. The pH-dependent changes in current amplitude could not be accounted for by the effects of altered surface charge. In recordings of unitary Na+ currents from outside-out membrane patches, acidification promoted channel opening to a reduced conductance level, near one-half of its maximal value. Acidification to pH < 6.0 also changed the kinetics of the current recruited with the lowest threshold from non-inactivating to inactivating, with the elimination of late openings. We conclude that lowering pH from an initial alkaline or neutral value blocks late Na+ current by reducing the number of contributing channels while also reducing the single channel conductance. The pH dependence of late Na+ current helps to explain clinically relevant changes in neuronal excitability in response to small (i.e. < 1 unit) perturbations in extracellular pH.

Animals↗

PubMed1999

Genetic differentiation of some Glossina morsitans morsitans populations.

To study the population structure of Glossina morsitans morsitans Westwood (Diptera: Glossinidae), polymerase chain reaction (PCR) and singlestrand conformational polymorphism (SSCP) methods were used to estimate mitochondrial DNA diversity at four loci in six natural populations from Zambia, Zimbabwe and Mozambique, and in two laboratory cultures. The Zambian and Zimbabwean samples were from a single fly belt. Four alleles were recorded at 12S and 16S1, and five alleles at 16S2 and COI. Nucleotide sequencing confirmed their singularities. Chi-square contingency tests showed that allele frequencies differed significantly among populations. Mean allele diversities in populations averaged over loci varied from 0.14 to 0.61. Little loss in haplotype diversity was detected in the laboratory cultures thereby indicating little inbreeding. Wright's fixation index F(ST) in the natural populations was 0.088+/-0.016, the correlation of haplotypes within populations relative to correlations in the total. A function of its inverse allows an estimate of the mean equivalent number of females exchanged per population per generation, 5.2. No correlation was detected between pairwise genetic distance measures and geographical distances. Drift explains the high degree of differentiation.

Africa, Southern↗

PubMed1999

Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting. In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp. The hDNA was efficiently repaired prior to DNA replication. The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.

Animals↗

PubMed1999

The molecular basis of multiple vector insertion by gene targeting in mammalian cells.

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.

Animals↗

PubMed1999

Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.

Animals↗

PubMed1999

The efficacy of routine outpatient management without antibiotics of fever in selected infants.

BACKGROUND: A previous study produced a protocol for outpatient management of febrile infants (FIs) judged to be at low risk for serious bacterial illness (SBI). This Philadelphia protocol demonstrated that 40% of FIs seen in the emergency department could be safely managed without antibiotics at home; and it was established by the emergency department staff as the standard of care at our institution. OBJECTIVE: To determine 1) the actual practices of management of FIs 18 months after establishment of the Philadelphia protocol as the standard of care, and 2) the continued efficacy of noninvasive outpatient management of fever in FIs who, using the Philadelphia protocol, were identified as low risk for SBI. DESIGN: Thirty-six-month consecutive cohort study. SETTING: Urban pediatric emergency department. PARTICIPANTS: Four hundred twenty-two infants, 29 to 60 days of age, with rectal temperatures >/=38.0 degrees C. Interventions. After a complete history taking, physical examination, and workup for SBI, infants were managed at the discretion of the attending physician in the emergency department. Subsequently, those management practices were reviewed and compliance with the Philadelphia protocol was evaluated. In addition, the overall efficacy and safety of that standard during 8 years of use was assessed. RESULTS: Of the 422 FIs enrolled, 101 (24%) were prospectively identified as low risk for SBI, and safe for management without antibiotics. Twenty-eight (6%) FIs were managed out of accordance with the Philadelphia protocol. Seven were admitted out of accordance, 10 (2 with UTI) were discharged out of accordance, and 11 inpatients (1 with bacterial gastroenteritis) initially received no antibiotics out of accordance with the protocol. Physician failure to consider the results of the complete blood count or urinalysis accounted for errors involving FIs with SBI. None of the 43 FIs with SBI were identified by the Philadelphia protocol to be at low risk for SBI. CONCLUSIONS: The Philadelphia protocol for outpatient management without antibiotics of FIs at low risk for SBI remains practical, reliable, and safe. Because breaches do occur, physicians must carefully scrutinize protocol compliance, especially with regard to the complete blood count and urinalysis.

Ambulatory Care↗