Biomedical subjects

K P Williams

Publications and source records attributed to K P Williams.

At least 19 recordsLinked to original sources

PubMed2001

Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway.

In the vertebrate embryo, neural cell types are organized spatially along the dorsoventral axis of the neural tube and differ by expression of cell-intrinsic determinants and by their adhesive and locomotory properties. Thus, dorsally, neural crest cells (NCC) show a strong propensity to disperse and migrate, whereas cells situated ventrally are highly cohesive and poorly motile. Members of the bone morphogenetic proteins have been shown to exert a dual role in the specification of dorsal neuroepithelial cells and in the dispersion of NCCs. To test whether Sonic hedgehog (Shh), another signaling molecule involved in the patterning of the ventral neural tube, might also contribute to the control of the adhesive and migratory potential of neuroepithelial cells, we analyzed the effect of ectopic Shh on NCC dispersion from neural tube explants cultured in vitro. The addition of Shh to the migration substrate of NCC caused inhibition of their dispersion. The effect of Shh on cell migration was reversible and was not accounted for by alterations of the specification, delamination, proliferation, and survival of NCCs but could be essentially attributed to a decreased cell-substrate adhesion mediated by integrins. In addition, Shh activity on cell migration was mediated by a specific N-terminal region of the molecule and was independent from the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Our study therefore reveals an unanticipated role for Shh in regulating adhesion and migration of neuroepithelial cells that is discernable from its inductive, mitogenic, and trophic functions.

Animals↗

PubMed2001

Fucosylation of Cripto is required for its ability to facilitate nodal signaling.

O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors. A single CR point mutation, Thr-88 --> Ala, results in a form of the protein that is not fucosylated and has substantially weaker activity in cell-based CR/Nodal signaling assays, indicating that fucosylation is functionally important for CR to facilitate Nodal signaling.

Amino Acid Sequence↗

PubMed2001

Enhanced potency of human Sonic hedgehog by hydrophobic modification.

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.

Acyl Coenzyme A↗

PubMed2001

Comparative biological responses to human Sonic, Indian, and Desert hedgehog.

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

Alkaline Phosphatase↗

PubMed2001

Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation.

A pool of stem cells that arise from the mesoderm during embryogenesis initiates hematopoiesis. However, factors that regulate the expansion of blood stem cells are poorly understood. We show here that cytokine-induced proliferation of primitive human hematopoietic cells could be inhibited with antibodies to hedgehog (Hh). Conversely, Sonic hedgehog (Shh) treatment induced the expansion of pluripotent human hematopoietic repopulating cells detected in immunodeficient mice. Noggin, a specific inhibitor of bone morphogenetic protein 4 (BMP-4), was capable of inhibiting Shh-induced proliferation in a similar manner to anti-Hh; however, anti-Hh had no effect on BMP-4-induced proliferation. Our study shows that Shh functions as a regulator of primitive hematopoietic cells via mechanisms that are dependent on downstream BMP signals.

Base Sequence↗

PubMed2001

Abdominal circumference: a single measurement versus growth rate in the prediction of intrapartum Cesarean section for fetal distress.

OBJECTIVE: The fetal abdominal circumference is the most sensitive ultrasound biometric measurement for predicting intrauterine growth restriction, which is associated with an increased risk of intrapartum fetal distress. We sought to evaluate and compare whether a third-trimester ultrasound measurement of abdominal circumference made within 1 week prior to delivery better predicts operative delivery for fetal distress when compared with the growth velocity of the abdominal circumference in the third trimester. METHODS: Retrospective analysis was carried out of prospectively collected ultrasound data on 117 patients with singleton gestations who had had at least two ultrasound assessments performed less than 6 weeks apart in the third trimester, with the last ultrasound performed within 1 week prior to delivery. The abdominal circumference value of the last ultrasound prior to delivery was placed into one of three categories: < or = 5% centile, > 5 to < or = 10% centile and > 10% centile for gestational age. The growth velocity of the abdominal circumference per week was placed into one of three categories: < or = 5 mm/week, 6-10 mm/week and > or = 11 mm/week. The chi-squared test was used to compare differences between the incidence of fetal distress between the groups. RESULTS: The incidences of Cesarean section for fetal distress in relation to a single measurement of the abdominal circumference were: < or = 5% centile, 8/23 (35%); > 5 to < or = 10% centile, 3/12 (25%); > 10% centile, 8/81 (10%) ( P < 0.05). The incidences of Cesarean section for fetal distress with the three abdominal circumference growth velocities were: < or = 5 mm/week, 9/55 (16%); 6-10 mm/week, 4/11 (36%); > or = 11 mm/week, 8/51 (16%) ( P = 0.9401). CONCLUSION: A single measure of the fetal abdominal circumference made within 1 week prior to delivery is superior to an assessment of growth rate of the fetal abdomen in the third trimester in discriminating patients who require Cesarean section for fetal distress.

Abdomen↗

PubMed2001

N-terminal fatty-acylation of sonic hedgehog enhances the induction of rodent ventral forebrain neurons.

The adult basal ganglia arise from the medial and lateral ganglionic eminences, morphologically distinct structures found in the embryonic telencephalon. We have previously shown that temporal changes in sonic hedgehog (Shh) responsiveness determine the sequential induction of embryonic neurons that populate the medial and lateral ganglionic eminences. In this report, we show that Shh-mediated differentiation of neurons that populate the lateral ganglionic eminence express different combinations of the homeobox-containing transcription factors Dlx, Mash1 and Islet 1/2. Furthermore, we show that N-terminal fatty-acylation of Shh significantly enhances its ability to induce the differentiation of rat E11 telencephalic neurons expressing Dlx, Islet 1/2 or Mash1. Recent evidence indicates that in utero injection of the E9.5 mouse forebrain with retroviruses encoding wild-type Shh induces the ectopic expression of Dlx2 and severe deformities in the brain. In this report, we show that Shh containing a mutation at the site of acylation prevents either of these phenotypes. These results suggest that N-terminal fatty-acylation of Shh may play an important role in Shh-dependent signaling during rodent ventral forebrain formation.

Animals↗

PubMed2000

tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter.

A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis. We have identified a circularly permuted version of the tmRNA gene in alpha-proteobacteria as well as in a lineage of cyanobacteria. The genes in these two groups seem to have arisen from two independent permutation events. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained. Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the alpha-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA.

Alphaproteobacteria↗

PubMed2000

Mapping sonic hedgehog-receptor interactions by steric interference.

We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of the protein were chosen for these studies and mutated to cysteine residues. These cysteines were then selectively modified with thiol-specific probes, and the modified proteins were tested for hedgehog receptor binding activity and their ability to induce differentiation of C3H10T1/2 cells into osteoblasts. Based on these analyses, approximately one-third of the Shh surface can be modified without effect on function regardless of the size of the attachment. These sites are located near to where the C terminus protrudes from the surface of the protein. All other sites were sensitive to modification, indicating that the interaction of Shh with its primary receptor Ptc is mediated over a large surface of the Shh protein. For sites Asn-50 and Ser-156, function was lost with the smallest of the probes tested, indicating that these residues are in close proximity to the Ptc-binding site. The epitope for the neutralizing mAb 5E1 mapped to a close but distinct region of the structure. The structure-activity data provide a unique view of the interactions between Shh and Ptc that is not readily attainable by conventional mapping strategies.

Antibodies, Monoclonal↗

PubMed2000

The tmRNA website.

The tmRNA Website collects all available tmRNA sequences into a single public resource, along with alignments and a guide to searching for new sequences. Over the last year, several sequences have been updated or newly found by monitoring ongoing genome sequencing projects; tmRNA sequence data from 70 species are now available. New features include: color-coding of sequences to mark suggested base-paired regions, a list of the literature concerning tmRNA, careful crediting of tmRNA sequence identifications, and a split browser window. Updates are very frequent. The tmRNA Website has a new URL: http:www.indiana.edu/tmrna

Internet↗

PubMed2000

Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration.

Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have been shown to be essential for the development of various organ systems, including neural, somite, limb, skeletal, and for male gonad morphogenesis. Sonic hedgehog and its cognate receptor Patched are expressed in the epithelial and/or mesenchymal cell components of the hair follicle. Recent studies have demonstrated an essential role for this pathway in hair development in the skin of Sonic hedgehog null embryos. We have further explored the role of the hedgehog pathway using anti-hedgehog blocking monoclonal antibodies to treat pregnant mice at different stages of gestation and have generated viable offspring that lack body coat hair. Histologic analysis revealed the presence of ectodermal placode and primodium of dermal papilla in these mice, yet the subsequent hair shaft formation was inhibited. In contrast, the vibrissae (whisker) development appears to be unaffected upon anti-hedgehog blocking monoclonal antibody treatment. Strikingly, inhibition of body coat hair morphogenesis also was observed in mice treated postnatally with anti-hedgehog monoclonal antibody during the growing (anagen) phase of the hair cycle. The hairless phenotype was reversible upon suspension of monoclonal antibody treatment. Taken together, our results underscore a direct role of the Sonic hedgehog signaling pathway in embryonic hair follicle development as well as in subsequent hair cycles in young and adult mice. Our system of generating an inducible and reversible hairless phenotype by anti-hedgehog monoclonal antibody treatment will be valuable for studying the regulation and mechanism of hair regeneration.

Animals↗

PubMed2000

Descriptive epidemiology of asthma in Trinidad, West Indies.

This cross-sectional study documents the demographic features of asthmatics seeking acute asthma care at four institutions on the island of Trinidad from January 1 to December 31, 1997. More than 15,000 (15,035) different patients made 27,353 visits during the study period. Pediatric and adolescent male patients outnumbered females, but this was reversed in patients aged 20-69 years. Frequency of visits per patient ranged from 1 to 242. Significant variation in monthly admissions was observed; admission rates were highest in the last quarter of the year but were lowest in July and August. The overall crude prevalence rate was estimated at 1.71 per 100 (range 1.16-2.34).

Adolescent↗

PubMed2000

A retrospective analysis of invasive Streptococcus pneumoniae infections in Trinidad.

A retrospective analysis of culture-positive cases of S pneumoniae from normally sterile body fluids is reported. Over 40% of patients were 5 years old or less while 28% of patients were 50 years old or more. Meningitis (44%) was the commonest clinical presentation followed closely by pneumonia (31%). The commonest predisposing disorder was human immunodeficiency virus infection though there were no identifiable risk factors in the majority of patients. Mortality from invasive pneumococcal disease was significantly higher in elderly patients compared with other age groups (p = 0.0003). In this study, all S pneumoniae isolates, for which there were antibiotic sensitivity data, were penicillin and/or amoxycillin sensitive.

Adolescent↗

PubMed1999

Zinc-dependent structural stability of human Sonic hedgehog.

The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K(d) values of approximately 1.6 and approximately 15 nM, respectively, as compared to a value of </=100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophysical and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biological function.

Alkaline Phosphatase↗

PubMed1999

Resuming translation on tmRNA: a unique mode of determining a reading frame.

The bacterial ribosome switches from an mRNA lacking an in-frame stop codon and resumes translation on a specialized RNA known as tmRNA, SsrA or 10Sa RNA. We find that the ribosome can reach and use the extreme 3' terminal codon of the defective mRNA prior to switching. The first triplet to be translated in tmRNA (the resume codon) is determined at two levels: distant elements in tmRNA restrict resume codon choice to a narrow window and local upstream elements provide precision. Insights from a randomization-selection experiment secure the alignment of tmRNA sequences from diverse species. The triplet UA(A/G) (normally recognized as a stop codon by release factor-1) is strongly conserved two nucleotides upstream of the resume codon. The central adenosine of this triplet is essential for tmRNA activity. The reading frame of tmRNA is determined differently from all other known reading frames in that the first translated codon is not specified by a particular tRNA anticodon.

Amino Acid Sequence↗

PubMed1999

The tmRNA website.

tmRNA (10Sa RNA) has a central role in trans -translation, in which a peptide tag encoded in tmRNA is added to the abnormally short protein product of a broken mRNA, as a signal for proteolysis of the entire tagged protein. The tmRNA website was established in 1997 as a resource for phylogenetic considerations of tmRNA structure and function. Since then, three partial tmRNA sequences have been completed, and sequences from 13 more species have been identified. Forty-six species from 10 bacterial phyla and chloroplasts are now represented in the database. Provisional sequence alignments and predicted proteolysis tag sequences are provided, as well as a literature review and a guide to searching for new tmRNA sequences. The tmRNA website is accessible via WWW at a new URL: http://sunflower.bio.indiana.edu/kwilliam/tmRNA /home.html

Bacteria↗

PubMed1999

Persistence of cerebral hemodynamic changes in patients with eclampsia: A report of three cases.

OBJECTIVE: This study was undertaken to compare the persistence of changes in estimated cerebral perfusion pressure and the resistance area product, an index of cerebrovascular resistance, in women with preeclampsia and women with eclampsia. STUDY DESIGN: The maternal middle cerebral artery was evaluated by transcranial Doppler ultrasonography in 6 patients with severe preeclampsia and 3 women with eclampsia, and cerebral blood flow velocities were determined. Estimated cerebral perfusion pressure was calculated according to the following equation: Estimated cerebral perfusion pressure = Mean cerebral blood flow velocity/(Mean cerebral blood flow velocity - Diastolic cerebral blood flow velocity) x (Mean blood pressure - Diastolic blood pressure). Because the diameters of the vessels could not be measured directly, an index of resistance was calculated according to the following equation: Resistance area product = Mean blood pressure/Mean cerebral blood flow velocity. We calculated an index of cerebral blood flow according to the following equation: Index = Estimated cerebral perfusion pressure/Resistance area product. Patients were reassessed at 24 and 48 hours after delivery. RESULTS: At the initial assessment the estimated cerebral perfusion pressure was higher in women with eclampsia than in those with severe preeclampsia. In addition, cerebrovascular resistance was significantly decreased in the eclampsia group, and it remained decreased as long as 4 days in 1 patient with eclampsia. CONCLUSIONS: Preeclampsia is associated with increases in cerebral perfusion pressure counterbalanced by increases in cerebrovascular resistance, with no change in cerebral blood flow. In eclampsia a significant increase in cerebral perfusion pressure occurs, but a significant fall in cerebrovascular resistance also occurs and a loss of autoregulation results in cerebral overperfusion similar to that of hypertensive encephalopathy. This may persist for as long as 4 days.

Cerebrovascular Circulation↗

PubMed1999

Evaluation of cerebral perfusion pressure changes in laboring women: effects of epidural anesthesia.

OBJECTIVE: To compare the effect of epidural anesthesia on cerebral perfusion pressure in laboring women. STUDY DESIGN: Maternal cerebral blood flow velocity was assessed in seven laboring patients with continuous epidural anesthesia and 15 without, using transcranial Doppler. Maternal cerebral blood flow velocity was assessed during the first stage at the trough of a contraction, at the peak of a contraction and at the second stage during pushing over the course of four contractions. Calculated estimated cerebral perfusion pressure: eCPP = Vmean/(Vmean - Vdiastolic) x (mean BP - diastolic BP), where V is velocity and BP is blood pressure; modified from Aaslid and colleagues. An index of cerebrovascular resistance, the resistance area product, was calculated: RAP = mean BP/mean velocity. We calculated an index of cerebral blood flow (cerebral blood flow index): CBF index = eCPP/RAP. RESULTS: In non-epidural patients, the eCPP fell significantly at the peak of a contraction and during pushing. Cerebrovascular resistance, RAP, rose significantly during the peak of a contraction, although cerebral blood flow did not change. In patients undergoing epidural anesthesia, the stages of labor had no significant effect on eCPP or RAP; however, these values were lower than those in patients without epidural anesthesia. CONCLUSIONS: The epidural group had a lower eCPP and RAP and cerebral blood flow index compared to the non-epidural group. In the non-epidural group, the mean arterial pressure was higher in all stages of labor with a trend towards an increase in eCPP and cerebral blood flow index.

Adult↗