Biomedical subjects
Publications and source records attributed to M Howard.
During her third pregnancy, a woman becomes preoccupied with illness and death. When the dysphoria continues after her child is born, how would you evaluate and manage the problem?
When this woman's long-standing PMS grows progressively more severe over the 3 years following the birth of her third child, what pharmacologic and nonpharmacologic treatments would you recommend? Symptoms of mood swings, irritability, and anxiety occur in many women during the premenstrual phase of the menstrual cycle. Several promising treatment options now exist for women whose symptoms are severe and interfere with daily functioning. These include nonpharmacologic as well as pharmacologic interventions, such as serotonergic antidepressants, anxiolytics, and hormones that suppress ovulation. When PMS becomes intolerably severe for this 36-year-old mother of 3 children--all under 10 years of age--she seeks treatment.
It has been demonstrated previously that respiratory secretions contain three oligomeric, gel-forming mucins; one of these was identified as the product of the MUC5AC gene (1). Here we demonstrate that the other two mucins are glycoforms of the MUC5B gene product. This was accomplished by trypsin treatment of the purified reduced mucin subunit populations and N-terminal sequencing of the liberated peptides. The products of trypsin digestion were separated by gel filtration into high molecular weight mucin glycopeptides and low molecular weight tryptic peptides. The latter were fractionated by reverse phase chromatography, and four of the major peptides were sequenced. Three of these peptides were identical to and contiguous within a 51-amino acid sequence deduced from a cDNA clone (JER57) encoding a portion of the MUC5B mucin. The other peptide is also present within this sequence but showed identity in only 9 of its 10 residues. A polyclonal antiserum raised against one of these peptides was reactive with the two putative MUC5B glycoforms. Analysis of the high molecular weight glycopeptides indicated that the MUC5B subunit contained different types and lengths of glycosylated domains; one domain of Mr 7.3 x 10(5), two domains of Mr 5.2 x 10(5), and a third domain of Mr 2.0 x 10(5). The amino acid composition of the larger two glycopeptides was similar in serine, threonine, and proline content but distinct from that of the smallest glycopeptide. Each of these domains in the mucin subunit is separated by a trypsin-sensitive region, and the relative abundance of the major peptides derived by proteolysis of these regions and their occurrence in a contiguous sequence suggest that they contain a common cysteine-rich motif.
This report shows, for the first time using modern analytical techniques, highly significant age-related decreases in chromium levels in 51,665 hair, sweat, and serum samples obtained from 40,872 patients referred by their physicians to an independent medical research clinic and laboratory (r = -.598 to -.762, P < .0001 for all correlations). Males were found to have significantly lower mean chromium levels than females (P < .05 to .0001). There was good correlation between chromium levels in hair, sweat, and serum (r = .536 to .729, P < .0001 for all correlations), indicating that hair and sweat chromium levels are valid additions to the serum levels in assessing chromium status. Chromium measurements in sweat, hair, and serum were performed using graphite furnace atomic absorption spectrophotometry. The influences that age-related decreases in chromium levels might have on increasing the risk to develop age-related impaired glucose metabolism, disordered lipid metabolism, coronary heart disease, arteriosclerosis, and type II diabetes mellitus are outlined, and the role that refined carbohydrates play in the development of compromised chromium status is presented.
Interleukin-7 (IL-7)-deficient mice exhibit an early defect in lymphopoiesis. We examined Bcl-2 expression and the cell cycle status of immature thymocyte subsets in these mice. In IL-7-deficient mice, developmental transition to a T cell-committed fate was accompanied by a striking loss of Bcl-2 protein expression and an increased relative proportion of cells in the G0/G1 stage of the cell cycle. Short-term culture of immature thymocytes with rIL-7 caused up-regulation of Bcl-2 protein and cell survival. These data specify a T cell lineage developmental transition point, prior to T cell antigen receptor rearrangement, where IL-7 signal transduction is linked to an anti-apoptosis mechanism and the cell cycle.
Post-transplant lymphoproliferative disorder (PTLD) is a well-recognized complication of organ transplant and has been associated with high mortality using conventional chemotherapy. We have investigated 11 cases of PTLD for alterations to the interferon alpha (IFNA) and p16 genes on chromosome 9p using archival material. 4/9 (44%) cases had deletions of the IFNA genes, in contrast to 1/59 (1.7%) cases of intermediate/high-grade de novo NHL drawn from the same geographical region. PTLD may therefore represent a distinct NHL subgroup exhibiting distinct gene pathology.
The germinal center (GC) develops in secondary lymphoid tissues in response to thymus-dependent (TD) antigens. To investigate the molecular mechanism of B cell differentiation in GC, we enriched GC B cells from spleen of TD antigen-immunized wild-type and X-linked immunodeficient (XID) mice, and examined the differentiation of GC B cells into antigen-specific IgG1 antibody-forming cells (AFC) in response to anti-CD40 mAb and cytokines. A significant proportion of freshly purified GC B cells expressed receptors for IL-4 and IL-5. Anti-CD40 mAb sustained the viability of GC B cells and IL-4 co-operated with anti-CD40 mAb for further enhancement of the cell viability. Anti-CD40 mAb and IL-4 were essential for inducing differentiation of GC B cells into antigen-specific IgG1-AFC and IL-5 efficiently enhanced their differentiation. GC B cells with the xid mutation responded for proliferation to CD40 ligation to a lesser extent and for the IgG1-AFC response to anti-CD40 mAb together with IL-4, but they showed impaired responsiveness to IL-5, regardless of enhanced expression of IL-5R in response to anti-CD40 mAb and IL-4. These results suggest that anti-CD40 mAb, IL-4 and IL-5 play a critical role in the differentiation of mouse GC B cells. The GC B cells from XID mice show a functional defect with respect to IL-5-mediated differentiation.
We asked whether inclusion of the FLAG epitope in the fourth extracellular loop of the cystic fibrosis transmembrane conductance regulator (M2-901/CFTR), which permits detection of cell surface expression, affected CFTR's biophysical properties or channel regulation by kinases, phosphatases, and nucleotides. Channel activity of M2-901/CFTR was evaluated in numerous cell types and expression systems to characterize its gating and regulation. Our results show that M2-901/CFTR required adenosine 3',5'-cyclic monophosphate-dependent protein kinase phosphorylation to initiate channel activity. Subsequently, ATP alone was sufficient to support channel gating, and ADP inhibited channel opening. Current fluctuation analysis indicated that the nucleotide-dependent gating rates were indistinguishable from those of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl- (11.2 pS), anion permeability ratio (1.66), and block by gluconate indicate that the anion conduction pathway is indistinguishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhibited M2-901/ CFTR channel activity by an identical mechanism to that described for wtCFTR. Finally, CFTR-dependent insertion and retrieval of cell membrane was unaffected by the presence of the FLAG epitope. These results indicate that this structural alteration does not affect the control mechanisms for channel gating and suggest that the fourth extracellular loop of CFTR does not contribute to the ion pore. Detection of M2-901/CFTR by a commercially available monoclonal antibody (M2), together with presentation of normal functional properties, makes M2-901/CFTR a valuable tool to evaluate CFTR protein expression and cellular location.
Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in cell-free extracts of Xenopus eggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they do in vivo, with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G1 variability in living cells is presented.
At least eight mucin apoproteins are expressed by the tracheobronchial epithelium, but it is not known which, if any, of these are major constituents of the respiratory secretions responsible for the formation of the mucus gel. To address this we have isolated mucins from normal, asthmatic and chronic bronchitic secretions. The asthmatic mucin reduced subunits were fractionated into four populations (I-IV) by anion-exchange HPLC. Amino acid and monosaccharide compositional analysis, as well as M(r) and size measurements, indicate that two of these populations (I and II) are glycoforms of the same or related apoprotein(s) and that the other populations contain two different apoproteins. A panel of antibodies and antisera recognizing the variable number tandem repeat (VNTR) of specific mucin apoproteins did not, as predicted, react with the glycosylated molecules, but after deglycosylation the majority of these probes (with the exception of those to MUC2, which were negative) reacted at a low level with each of the subunit populations. In contrast, an antiserum against a non-VNTR sequence of MUC5AC identified one of the populations (III) as the MUC5AC mucin. The MUC5AC reduced subunit had an M(r) of 2.2 x 10(6) and an RG (radius of gyration) of 57 nm. The genetic identities of the major mucin (populations I and II) and a minor component (population IV) were not established. The MUC5AC mucin was also identified as a major component in the pooled normal secretions from 20 individuals, whereas in a chronic bronchitic sample it was only a minor constituent. Furthermore, in all these different respiratory secretions the MUC5AC mucin appears as a similar biochemical entity, as assessed by Mono Q chromatography and agarose electrophoresis, suggesting that it may have a well-defined pattern of glycosylation in the respiratory tract.
The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5 x 10(6) to 40 x 10(6) Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873-880].
We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up-regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti-IgM and interleukin-4 (IL-4) alone did not induce significant Fas expression but enhanced CD40L-mediated up-regulation of Fas expression. The T cell-derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas-mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas-mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas-mediated death signal was observed in 2-day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas-mediated apoptosis than were cells stimulated with CD40L plus anti-IgM or IL-4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co-stimulation of B cells in the presence of CD40L and anti-Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.
STUDY OBJECTIVE: To define the contribution of domestic violence (DV) to homicides in women in New Mexico and to examine differences in ethnicity, mechanism, previous documented injuries, incidence of sexual assault, and use of alcohol or illicit drugs between DV- and non-DV-related homicides. METHODS: We carried out a retrospective analysis of reports of the state office of the medical investigator (OMI) reports from all female homicides from 1990 to 1993 in New Mexico. A homicide was defined as being related to DV if the perpetrator was a current or former male intimate partner. The chi-squared and Mann-Whitney tests were used to analyze data. RESULTS: The OMI investigated 134 homicides in women for an overall fatality rate of 4.3 per 100,000. A male intimate partner was the perpetrator in 62 cases (46%). The rate of DV homicide among American Indians (4.9 per 100,000) was significantly higher than that among Hispanics (1.7) and non-Hispanic whites (1.8)(RR=2.8; 95% confidence interval (CI), 1.5 to 5.1). Firearms were almost two times as likely to be used in DV homicides as in non-DV homicides (RR=1.8; 95% CI, 1.2 to 2.6). Evidence of old injuries was found more often in DV homicide cases (35.5%) than in non-DV cases (83%) (RR=4.3; 95% CI, 1.8 to 9.8). The presence of alcohol or other drugs was higher among non-DV homicide victims (69%) than DV homicide victims (54.3%) (P=.03). CONCLUSION: American Indian women are at particularly high risk of homicide, including DV homicide. Firearms were overrepresented in DV homicides, suggesting that removing firearms from the homes of previous DV perpetrators would be a useful public health strategy. Alcohol or illicit drugs were found in approximately two thirds of New Mexico women who were victims of homicide. The high prevalence of history of previous injuries among DV homicide victims indicates that early identification of DV victims in the emergency department and other health care settings is an important point of intervention.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel responsible for cAMP-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection in membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. The function of six different tagged-CFTRs was tested in two different physiological assays. CFTRs containing the M2 epitope responded to cAMP, whereas cells expressing CFTR with the hemagglutinin HA tag showed little or no cAMP response. Using CFTR tagged in the fourth external loop, we demonstrate that cAMP activation using forskolin results in an increase in CFTR in the plasma membrane of HeLa cells. Forskolin inhibited CFTR endocytosis, and this contributes to the increase in cell surface CFTR expression. Our results indicate that regulation of cell surface CFTR contributes to the increase in plasma membrane Cl conductance evoked by cAMP stimulation.
Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (deltaF508), causes severe CF and resides on 70% of mutant chromosomes. Severe CF is also caused by premature stop mutations, which are found on 5% of CF chromosomes. Here we report that two common, disease-associated stop mutations can be suppressed by treating cells with low doses of the aminoglycoside antibiotic G-418. Aminoglycoside treatment resulted in the expression of full-length CFTR and restored its cyclic AMP-activated chloride channel activity. Another aminoglycoside, gentamicin, also promoted the expression of full-length CFTR. These results suggest that treatment with aminoglycosides may provide a means of restoring CFTR function in patients with this class of mutation.
A 14-year-old girl with HIV infection and subcutaneous Kaposi's sarcoma (KS) received thalidomide therapy for oral ulcers, resulting in regression of KS lesions, disappearance of KS-associated herpesvirus (KSHV) DNA from blood, and reduced viral load in tumor tissue. Administration of grauntocyte colony-stimulating factor resulted in clinical exacerbation of KS and reappearance of KSHV DNA in blood.
OCT embedded cryostat sections of stored pathological specimens of non-Hodgkin's lymphoma were used to provide RNA. After reverse transcription to produce cDNA, the polymerase chain reaction was performed with primers for standard and variant forms of the CD44 molecule. Using Southern transfer and hybridisation with a probe specific for exon 4 of the CD44 gene, both standard and variant forms were visualised by autoradiography. This method was shown to be applicable to other gene products by using primers specific for the abl and bcr genes. This technique permits retrospective analysis of RNA from small amounts of stored pathological samples.
We examined the relation between Cl current (Icl) stimulation and cell membrane capacitance (Cm) when cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in Xenopus oocytes. ICl and Cm increased in parallel when oocytes expressing CFTR were stimulated by forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM). The adenosine 3',5'-cyclic monophosphate (cAMP)-induced increase in surface area detected by Cm was confirmed by morphometry in the same oocytes used for the electrical recordings. These increases in ICl and Cm were reversible and were absent from control oocytes not injected with CFTR cRNA. The time to reach peak ICl lagged slightly behind the peak in Cm. ICl was varied by altering CFTR expression level or agonist dose or by expressing different CFTR mutants. In all cases, there was a close correlation between ICl and Cm, and the kinetics of ICl and Cm stimulation were more rapid the larger the magnitude of the stimulated current. The Cm-ICl relation for wild-type CFTR saturated, consistent with a limited capacity of cells to increase their surface area. These results indicate that stimulation of the CFTR ICl is linked closely to increases in membrane area. This suggests that CFTR is present in the membrane vesicles whose insertion is stimulated by cAMP. The contents of these vesicles may provide a link between activation of CFTR and its cAMP-dependent regulation of other channels.