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Biomedical subjects

H Lam

Publications and source records attributed to H Lam.

At least 37 records · Page 2Linked to original sources
PubMed1999

Genomic organization of the human hairless gene (HR) and identification of a mutation underlying congenital atrichia in an Arab Palestinian family.

Congenital atrichia is a rare form of hereditary human hair loss, characterized by the complete shedding of hair shortly after birth, together with the formation of papular lesions on the skin. Recently, we cloned the human homolog of the mouse hairless gene and identified pathogenic mutations in several families with inherited congenital atrichia. Here, we present the genomic organization of the human hairless gene (HGMW-approved symbol HR), which spans over 14 kb on chromosome 8p12 and is organized into 19 exons. In addition, we report the identification of a 22-bp deletion mutation in exon 3 of the hairless gene in a large consanguineous Arab Palestinian family from a village near Jerusalem, Israel. These findings extend the body of evidence implicating mutations in the hairless gene as an underlying cause of congenital atrichia in humans.

Alopecia↗
PubMed1999

High-frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein-mediated invasion and cytoskeletal rearrangements.

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90-226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti-M1 antibody, invasion by an M1- strain (90-226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper-like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal-containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP-1.

Actins↗
PubMed1999

Erythropoietic protoporphyria: identification of novel mutations in the ferrochelatase gene and comparison of biochemical markers versus molecular analysis as diagnostic strategies.

BACKGROUND: Erythropoietic protoporphyria (EPP) results from an inherited deficiency of the last enzyme of the heme biosynthetic pathway, ferrochelatase (FC). EPP is usually inherited in an autosomal dominant fashion, and the mutations in the FC gene on chromosome 18q21.3 detected in EPP patients are heterogeneous. METHODS: In this study, we screened the FC gene for mutations in 12 patients from 10 unrelated families with EPP and their family members using heteroduplex analysis, automated sequencing, and restriction enzyme digestion. RESULTS: We detected 8 different mutations in these patients, including 1 missense mutation, 5 frameshift mutations, and 2 splice site mutations, 6 of which are previously undescribed. CONCLUSIONS: We have established the molecular basis of EPP in 10 unrelated families, thereby providing further evidence for the heterogeneity in this disorder. Importantly, molecular diagnosis allowed revisions in the status of several clinically unaffected silent mutation carriers within the families. We compare the value of genetic research strategies with the combination of biochemical data and clinical phenotype as diagnostic tools to confirm a putative diagnosis in EPP.

Biomarkers↗
PubMed1999

A comparison of pregnancy outcome between high-order multiple and twin pregnancies: matched-pair retrospective study.

OBJECTIVE: To compare the pregnancy outcome between high-order multiple and twin pregnancies. DESIGN: Matched-pair retrospective analysis. SETTING: University teaching hospital, Hong Kong. PATIENTS: Patient records from 38 high-order multiple pregnancies that were delivered over a period of 15 years, and those from matched twin pregnancies. MAIN OUTCOME MEASURES: Obstetric and perinatal outcomes. RESULTS: The incidence of high-order multiple pregnancies increased over the study period in association with the more frequent practice of ovulation induction and other assisted reproductive techniques. High-order multiple pregnancies were associated with a higher incidence of maternal complications and a significantly higher perinatal mortality rate than were twin pregnancies. CONCLUSION: Efforts should be made to prevent multiple pregnancies by carefully monitoring ovulation treatment and by limiting the number of embryos transferred.

Journal Article↗
PubMed1998

EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

Amino Acid Sequence↗
PubMed1998

Production and characterization of SIV envelope-specific rhesus monoclonal antibodies from a macaque asymptomatically infected with a live SIV vaccine.

Five rhesus monoclonal antibodies (RhMAbs) were produced by rhesus EBV transformation of peripheral blood B cells from a rhesus macaque that had been asymptomatically infected with an attenuated, macrophage-tropic SIV strain, 17E-Cl. These MAbs recognized conformation-dependent epitopes on SIV gp120 and could not be mapped using synthetic peptides. All five RhMAbs were able to neutralize the vaccine strain and a heterologous isolate, SIV/DeltaB670. The RhMAbs did not cross-react with HIV-2; by contrast, four human MAbs derived from an HIV-2-infected person were broadly cross-reactive with both SIV and HIV-2 gp120s. Cross-competition analysis indicated that the five RhMAbs could be placed in two groups recognizing two nonoverlapping epitopes; while the HMAbs were placed in two additional competition groups. Binding of the three group I RhMAbs (1.7F, 3.11B, and 1.10A) as well as HMAb 17A was shown to be sensitive to specific amino acid alterations in V4 occurring in natural env variants. The results of this study demonstrate that RhEBV transformation provides a means to probe rhesus antibody responses to SIV infection at the monoclonal level. RhMAbs will allow structural and functional studies of envelope glycoprotein determinants that elicit protective immune responses against SIV.

Amino Acid Sequence↗
PubMed1998

Alopecia universalis associated with a mutation in the human hairless gene.

There are several forms of hereditary human hair loss, known collectively as alopecias, the molecular bases of which are entirely unknown. A kindred with a rare, recessively inherited type of alopecia universalis was used to search for a locus by homozygosity mapping, and linkage was established in a 6-centimorgan interval on chromosome 8p12 (the logarithm of the odds favoring linkage score was 6.19). The human homolog of a murine gene, hairless, was localized in this interval by radiation hybrid mapping, and a missense mutation was found in affected individuals. Human hairless encodes a putative single zinc finger transcription factor protein with restricted expression in the brain and skin.

Alopecia↗
PubMed1998

Quantitative immunolocalization of mu opioid receptors: regulation by naltrexone.

The present study utilized a newly developed quantitative immunohistochemical assay to measure changes in mu opioid receptor abundance following chronic administration of the opioid receptor antagonist naltrexone. These data were compared with those obtained from mu receptor radioligand binding on adjacent tissue sections, in order to determine whether the characteristic antagonist-induced increase in radioligand binding is due to an increase in the total number of mu receptors and/or to an increase in the proportion of receptors that are in an active binding conformation in the absence of a change in the total number of receptors. Adult male Sprague-Dawley rats were administered naltrexone, 7-8 mg/kg per day, or saline continuously for seven days by osmotic minipumps, after which time their brains were processed for immunohistochemistry and receptor autoradiography on adjacent fresh frozen tissue sections. Semiquantitative immunohistochemistry was performed using a radiolabelled secondary antibody for autoradiographic determination and a set of radioactive standards. Results demonstrate an overall concordance between the distribution of mu opioid receptors as measured by the two different methods with a few exceptions. Following naltrexone administration, mu receptor immunoreactivity was significantly higher in the amygdala, thalamus, hippocampus, and interpeduncular nucleus as compared with the saline-treated control animals. [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin binding to mu opioid receptors was significantly higher in the globus pallidus, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, ventral tegmental area, central gray, and interpeduncular nucleus of the naltrexone-treated rats. These findings indicate that in some brain regions chronic naltrexone exposure increases the total number of mu opioid receptors, while in other regions there is an increase in the percent of active receptors without an observable change in the total number of receptors. Quantitative receptor immunodetection together with ligand autoradiography provides a new approach for investigating the regulation of mu opioid receptors on tissue sections.

Analgesics, Opioid↗
PubMed1998

High-frequency intracellular infection and erythrogenic toxin A expression undergo phase variation in M1 group A streptococci.

A clonal variant of serotype M1 group A streptococcus, strain 90-131, disseminated to several continents, where it was associated with severe systemic infections and toxic shock. Although this strain harbours the speA gene and is efficiently internalized by human epithelial cells, clinical isolates often fail to express the erythrogenic toxin under laboratory growth conditions. Cultures of strain 90-131 were observed to phase vary between small, dry, compact and larger, more mucoid colonies. The former were shown to be poorly internalized by epithelial cells. Analysis of RNA by Northern hybridization demonstrated that the emml, hasA and speA genes were weakly transcribed in cultures derived from the small colonies and highly transcribed in those derived from the large colonies. An insertion mutation in mga (the multigene activator) downregulated the invasion of epithelial cells and the transcription of emm1 and hasA, but had little impact on the transcription of speA. These are the first data to suggest the existence of a common regulatory circuit linking intracellular invasion, M protein, hyaluronic acid capsule and erythrogenic toxin expression by group A streptococcus. Moreover, the genetic instability of toxin expression exhibited by this serotype may impact on laboratory studies that attempt to associate toxin production with toxic shock.

Antigens, Bacterial↗
PubMed1998

Homozygous variegate porphyria: identification of mutations on both alleles of the protoporphyrinogen oxidase gene in a severely affected proband.

Homozygous variegate porphyria is a severe skin and neurologic disease manifesting in early infancy, and characterized by markedly reduced levels of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase. We investigated the molecular basis of variegate porphyria, usually an autosomal dominantly inherited trait, in a severely affected female proband and her parents. The mutation detection strategy included heteroduplex analysis, automated sequencing, and allele specific oligonucleotide hybridization. We identified two underlying missense mutations in the protoporphyrinogen oxidase gene, consisting of a G-to-A transition in exon 6 (G169E), and a G-to-A transition in exon 10 (G358R). Our study establishes the molecular basis of "homozygous" variegate porphyria for the first time, in demonstrating that this patient is a compound heterozygote for two different missense mutations in the protoporphyrinogen oxidase gene.

Adult↗
PubMed1998

A missense mutation in the zinc-finger domain of the human hairless gene underlies congenital atrichia in a family of Irish travellers.

Congenital atrichia is a rare, recessively inherited form of hair loss affecting both males and females and is characterized by a complete absence of hair follicles. Recently, a mutation in the human hairless gene was implicated in the pathogenesis of congenital atrichia. The human hairless gene encodes a putative single zinc-finger transcription-factor protein with restricted expression in brain and skin, which is believed to regulate catagen remodeling in the hair cycle. In this study, we report the identification of a missense mutation in the zinc-finger domain of the hairless gene in a large inbred family of Irish Travellers with congenital atrichia. The mutated arginine residue is conserved among human, mouse, and rat, suggesting that it is of significant importance to the function of the zinc-finger domain.

Alopecia↗
PubMed1998

Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells.

The ability of a serotype M1 strain of Streptococcus pyogenes to efficiently invade A549 human lung epithelial cells was previously shown to be dependent on bacterial exposure to human or bovine serum proteins or synthetic peptides containing the sequence RGD. In this study, stimulation by invasion agonists was determined to be dependent on expression of the streptococcal cell surface protein, M1. Fetal bovine serum (FBS), fibronectin (Fn), the extracellular matrix protein laminin (Lm), and RGD-containing peptides were tested for their abilities to promote epithelial cell invasion and adherence by isogenic M1(+) and M1(-) strains of S. pyogenes. In the absence of an agonist, invasion and adherence were comparable for the two bacterial strains. FBS, Fn, and Lm stimulated invasion of the M1(+) strain as much as 70-fold but failed to significantly affect invasion by the M1(-) mutant. Adherence of the wild-type strain was stimulated by these same agonists. Epithelial cell adherence by the M1(-) strain, however, was unaffected by the presence of Fn or Lm. Several RGD-containing peptides were found to promote invasion independently of M1 expression. Binding of 125I-Fn was reduced 88% by the M1(-) mutation and Fn was found to bind purified M1 protein, suggesting that Fn mediates invasion by direct binding to M1. To determine if host integrins might be involved in internalization of streptococci, several anti-integrin monoclonal antibodies (MAbs) were tested for their abilities to inhibit invasion. Antibody directed against integrin beta1 inhibited FBS-, Fn-, and Lm-mediated invasion but did not abrogate RGD-peptide-stimulated invasion. MAb directed against the epithelial cell Fn receptor, integrin alpha5beta1, inhibited Fn and FBS-mediated invasion but did not specifically inhibit Lm-mediated invasion. These results indicate that S. pyogenes has evolved multiple mechanisms for invasion of eukaryotic cells, at least two of which involve interactions between M1 protein, host integrins, and integrin ligands.

Antigens, Bacterial↗
PubMed1998

A globally disseminated M1 subclone of group A streptococci differs from other subclones by 70 kilobases of prophage DNA and capacity for high-frequency intracellular invasion.

The M1inv+ subclone of M1 group A streptococci that spread globally in the late 1980s and early 1990s was previously identified by restriction fragment length polymorphism (RFLP), M protein, and SpeA exotoxin sequence analyses. Strains representing this subclone were characterized with regard to carriage of bacteriophage and capacity to invade cultured human epithelial cells. The M1inv+ subclone was found to harbor two entirely different prophages, phage T13 and phage T14, which together supplement its genome with nearly 70 kb of DNA. Phage T14 encodes the SpeA exotoxin and is closely related to the classic converting phage T12. Plaque-forming characteristics and RFLP analyses of phages T13 and T14 were compared to each other and to phage T12. Other subclones of M1, isolated in the 1970s to the early 1980s, lacked both prophages. The M1inv+ subclone was previously reported to be efficiently internalized by human epithelial cells. This potential was confirmed and expanded by comparing a variety of clinical isolates. The capacity for high-frequency invasion of epithelial cells was not transmitted to a laboratory strain of group A streptococci by the above-mentioned bacteriophages.

Clone Cells↗
PubMed1998

A complex control system for transcriptional activation from the sid promoter of bacteriophage P4.

The sid gene promoter (Psid), which controls expression of the late genes from satellite phage P4, is activated by a unique class of small DNA-binding proteins. The activators from both satellite and helper phages stimulate transcription from Psid. These activators bind to sites centered at position -55 in all the helper and satellite phage late promoters. P4 Psid is unique in that it has an additional activator binding site centered at position -18 (site II). We have constructed a mutant of site II that no longer binds activators. Transcription under the control of satellite phage activators is increased by the site II mutation. In contrast, helper phage activators do not show this increase in transcription from Psid mutated at site II. Competition gel shift analysis reveals that the P4 satellite phage activator, Delta, binds eightfold better to site II than to site I. The products of the sid transcription unit are needed only when a helper phage is present; thus, the satellite phage activators repress transcription until the helper is present to supply a nonrepressing activator.

Base Sequence↗
PubMed1998

Molecular basis of variegate porphyria: a missense mutation in the protoporphyrinogen oxidase gene.

Variegate porphyria (VP) is an autosomal dominant disorder characterised by a partial defect in the activity of protoporphyrinogen oxidase (PPO), and has recently been genetically linked to the PPO gene on chromosome 1q22-23 (Z=6.62). In this study, we identified a mutation in the PPO gene in a patient with VP and two unaffected family members. The mutation consisted of a previously unreported T to C transition in exon 13 of the PPO gene, resulting in the substitution of a polar serine by a non-polar proline (S450P). This serine residue is evolutionarily highly conserved in man, mouse, and Bacillus subtilis, attesting to the importance of this residue. Interestingly, the gene for Gardner's syndrome (FAP) also segregates in this family, independently of the VP mutation. Gardner's syndrome or familial adenomatous polyposis (FAP) is also an autosomal dominantly inherited genodermatosis, and typically presents with colorectal cancer in early adult life secondary to extensive adenomatous polyps of the colon. The specific gene on chromosome 5 that is the site of the mutation in this disorder is known as APC (adenomatous polyposis coli), and the gene has been genetically linked to the region of 5q22.

Adenomatous Polyposis Coli↗
PubMed1997

Molecular basis of variegate porphyria: a de novo insertion mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders that result from the inherited or acquired dysregulation of one of the eight enzymes in the heme biosynthetic pathway. Variegate porphyria (VP) is characterized by deficiencies in protoporphyrinogen oxidase (PPO) and has recently been genetically linked (Z = 6.62) to the PPO gene on chromosome 1q21. In this study, we have identified two sequence variants in the PPO gene in a family with VP. The first is a neutral polymorphism at the -47 position of intron 2; this polymorphism is present in the general population and is unlikely to underlie the VP phenotype. The second is a mutation in the PPO gene in a patient with VP; the mutation consists of an apparently de novo 2-bp insertion in exon 3 of PPO and results in a frameshift and downstream premature termination codon. These data establish that a frameshift mutation in PPO is the underlying mutation in this patient with VP and explain the sporadic occurrence of the phenotype in this family.

Adult↗