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Biomedical subjects

K Mack

Publications and source records attributed to K Mack.

At least 19 recordsLinked to original sources
PubMed2002

A mutant of Burkholderia pseudomallei, auxotrophic in the branched chain amino acid biosynthetic pathway, is attenuated and protective in a murine model of melioidosis.

Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.

Amino Acids, Branched-Chain↗
PubMed2002

Osteoblastic response to the defective matrix in the osteogenesis imperfecta murine (oim) mouse.

This work examines the cellular pathophysiology associated with the weakened bone matrix found in a murine model of osteogenesis imperfecta murine (oim). Histomorphometric analysis of oim/oim bone showed significantly diminished bone mass, and the osteoblast and osteoclast histomorphometric parameters were increased in the oim/oim mice, compared with wild-type (+/+) mice. To assess osteoblast activity, a rat Col1a1 promoter linked to the chloramphenicol acetyltransferase reporter transgene was bred into the oim model. At 8 d and 1 month of age, no difference in transgene activity between oim and control mice was observed. However, at 3 months of age, chloramphenicol acetyl transferase activity was elevated in oim/oim;Tg/Tg, compared with +/+;Tg/Tg and oim/+;Tg/Tg. High levels of urinary pyridinoline crosslinks in the oim/oim;Tg/Tg mice were present at all ages, reflecting continuing high bone resorption. Our data portray a state of ineffective osteogenesis in which the mutant mouse never accumulates a normal quantity of bone matrix. However, it is only after the completion of the rapid growth phase that the high activity of the oim/oim osteoblast can compensate for the high rate of bone resorption. This relationship between bone formation and resorption may explain why the severity of osteogenesis imperfecta decreases after puberty is completed. The ability to quantify high bone turnover and advantages of using a transgene that reflects osteoblast lineage activity make this a useful model for studying interventions designed to improve the bone strength in osteogenesis imperfecta.

Amino Acids↗
PubMed2000

The Internet and clinical practice of child neurology.

The Internet has great potential for aiding patient care. Concerns regarding patient confidentiality, provider liability, and efficient and fair use of these new resources have triggered specific recommendations for integration of the Internet into our practices.

Communication↗
PubMed1999

Neuropsychological and behavioral status of children with complex partial seizures.

Neuropsychological and behavioral status were examined in 57 children aged 7 to 16 years with complex partial seizures (CPS) and compared with 27 sibling control children of the same age. Epilepsy had a significant effect on both cognitive and behavioral adjustment measures. Children with CPS had significant impairment across all seven cognitive domains assessed, reflective of a profile of relatively diffuse and generalized cognitive dysfunction. Age at onset of recurrent seizures was the strongest and most consistent predictor of adequacy of cognitive functioning; earlier age at onset was associated with poorer cognitive status. Children with CPS also had more problems compared with sibling control children on measures of social and school competence and internalizing behavior problems, but not externalizing behaviors. Further, frequency of seizure activity in the past year, rather than age at seizure onset, emerged as the strongest predictor of these behavioral difficulties. These findings are discussed in the context of understanding the impact of CPS on cognition and behavioral adjustment, and identifying the contribution of various aspects of the neurodevelopmental course of CPS to these issues.

Achievement↗
PubMed1999

Schizophrenia, mental state, and mother-infant interaction: examining the relationship.

OBJECTIVE: The aim of this study is to examine the role that disturbance of mental state of hospitalised mothers with a postpartum schizophrenic illness plays in determining the quality of mother-infant interactions. METHOD: We examined the relationship between the nature and severity of symptomatology in mothers with schizophrenia and the quality of her interactions with her infant in a sample of 15 mother-infant dyads admitted to a psychiatric Mother-Baby Unit. Data were obtained at admission and discharge. RESULTS: Mothers with florid positive symptoms and prominent negative symptoms of schizophrenia and their infants were identified as being at particular risk of displaying disturbed interactions. The adverse contribution of negative symptoms was often not evident until after the positive symptoms had resolved. CONCLUSIONS: Given that negative symptoms are often treatment resistant, optimal care of mothers with schizophrenia and their infants needs to involve ongoing therapeutic intervention which specifically addresses disturbances of mother-infant interaction. Further research is required to identify which interventions are likely to be of greatest benefit.

Adult↗
PubMed1999

Quantification of sub-femtomole amounts of Alzheimer amyloid beta peptides.

We evaluated methods for the quantitative Western blot analysis of A beta 1-40 and A beta 1-42. Both chromogenic and chemiluminescent detection methods gave similar sensitivities (0.15 fmol of A beta 1-40 and 0.3 fmol of A beta 1-42); however, the chromogenic method was more rapid, simpler, less expensive and gave fewer background problems; consequently, it yielded more reliable results. Adsorption to various types of laboratory plasticware can greatly interfere with the accurate measurement of A beta, but this can be prevented by the addition of SDS or bovine serum albumin. Among several methods for concentrating A beta from biological materials, immunoadsorption to Sepharose-bound antibodies was the most efficient. It yielded 50% recovery of 1 pM A beta 1-42 or A beta 1-40 and so was a suitable method to measure A beta levels in human plasma. Through combined immunoadsorption and Western blotting we could determine the amounts of A beta isoforms secreted from 1 x 10(6) cells after a culture period as short as 1 h. This eliminates the need to use radiolabelling or over-expression to study A beta precursor processing Bovine serum contains subnanomolar A beta levels, similar to those that reportedly stimulate cell proliferation. That cultured cells quickly secrete these levels of A beta suggests that the peptide might exert an autocrine effect.

Alzheimer Disease↗
PubMed1998

The detection of insertion sequences within the human pathogen Burkholderia pseudomallei which have been identified previously in Burkholderia cepacia.

Using primers designed from the nucleotide sequences of five insertion elements identified previously in Burkholderia cepacia, the presence of two insertion sequences (IS406 and IS407) was detected in chromosomal DNA isolated from strains of the human pathogen Burkholderia pseudomallei. The IS407 homologue was cloned from B. pseudomallei NCTC 4845 and nucleotide sequenced to confirm its identity and degree of homology with B. cepacia IS407. A PCR amplification product from B. pseudomallei NCTC 4845 DNA provided an IS407 probe which was used to determine, by Southern blotting, the number and location of copies of IS407 in ten strains of B. pseudomallei and four representatives from three of the five genomovars of B. cepacia.

Base Sequence↗
PubMed1996

Transformation of Burkholderia pseudomallei by electroporation.

Conditions were defined for the successful transformation of the human pathogen Burkholderia pseudomallei 4845 by electroporation, using the incQ plasmid pKT230. We have obtained frequencies of up to 8 x 10(3) transformants per microgram plasmid DNA with freshly prepared electrocompetent B. pseudomallei. The method also allows for easy and reproducible production of frozen cell suspensions which can produce efficiencies up to 2.5 x 10(2) transformants per microgram of plasmid DNA. Kanamycin had to be added to the culture growth medium at a minimum concentration of 20 micrograms ml-1 and a maximum concentration of 50 micrograms ml-1 for the bacteria to become electrocompetent. Bacteria grown in medium containing a final concentration of 30 micrograms ml-1 kanamycin produced the highest numbers of transformants, although the transformation frequency at this concentration was not as efficient as that at 50 micrograms ml-1. Electron microscopy indicated that the kanamycin affects the integrity of the bacterial outer membrane, which becomes loose and wrinkled in appearance. The electrocompetence does not result in a permanent morphological change.

Burkholderia pseudomallei↗
PubMed1996

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

Animals↗
PubMed1995

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice.

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

Animals↗
PubMed1995

Usability of manual handling aids for transporting materials.

Manual transport aids (trucks and trolleys) are in widespread use throughout most industries, but their use does not always result in the anticipated reduction of workload or musculoskeletal stress. A survey of users has shown that many of the aids currently used are poorly designed or inappropriate for the tasks performed. The information gained during the survey has been analysed to identify the most important design features and to provide guidance for their selection and evaluation, in order to ensure that aids are suitable for the tasks for which they are used and that they are effective and safe. It is clear that the first stage in establishing design criteria and guidelines should be developing an understanding of the task requirements and environmental conditions under which materials have to be transported in industry.

Journal Article↗
PubMed1995

Differential regulation of COL2A1 expression in developing and mature chondrocytes.

To investigate the regulation of type II collagen gene expression in cells undergoing chondrogenic differentiation, we have employed a 5-kbp genomic fragment of the human type II collagen gene which contains 1.8kbp of upstream sequences, the transcription start site, the first exon and 3 kbp of intronic sequences, fused to either lac Z or chloramphenicol acetyl transferase-reporter gene. Transient expression studies revealed a parallel increase in transgene activity and endogenous type II collagen mRNA levels during the onset of the cartilage differentiation of limb mesenchymal cells in high-density micromass cultures. At later periods in culture, however, the transgene activity declines, although steady-state levels of type II collagen mRNA are reported to continue to increase (Kosher et al.: J. Cell. Biol. 102: 1151-1156, 1986; Kravis and Upholt. Dev. Biol. 108: 164-172, 1985). In addition, the activity of the transgene is seven-fold higher at the onset of chondrogenic differentiation in micromass cultures that in well differentiated sternal chondrocytes, although similar levels of type II collagen transcripts are found in these cells. Furthermore, deletions of intronic segments resulted in greater drop in activity of the constructs in differentiating chondrocytes in micromass cultures than in mature sternal chondrocytes. The expression of the construct in transgenic mice is higher at the onset of chondrogenic differentiation and in newly differentiated chondrocytes than in more mature differentiated chondrocytes. Based on these observations, it appears that the mechanisms involved in the regulation of the type II collagen gene at the onset of chondrocyte differentiation are different from those resulting in the maintenance of its expression in fully differentiated chondrocytes.

Animals↗
PubMed1995

Identification of regulatory elements necessary for the expression of the COL1A1 promoter in murine odontoblasts.

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.

Animals↗