Introduction of community-based skin-to-skin care in rural Uttar Pradesh, India.

OBJECTIVE: Two-thirds of women globally give birth at home, yet little data are available on use of skin-to-skin care (STSC) in the community. We describe the acceptability of STSC in rural Uttar Pradesh, India, and measured maternal, newborn, and ambient temperature in the home in order to inform strategies for introduction of STSC in the community. STUDY DESIGN: Community-based workers in intervention clusters implemented a community mobilization and behavior change communication program that promoted birth preparedness and essential newborn care, including adoption of STSC, with pregnant mothers, their families, and key influential community members. Acceptance of STSC was assessed through in-depth interviews and focus groups, and temperature was measured during home visits on day of life 0 or 1. RESULTS: Incidence of hypothermia (<36.5 degrees C) was high in both low birth weight (LBW) and normal birth weight (NBW) infants (49.2%, (361/733) and 43% (418/971), respectively). Mean body temperature of newborns was lower (P<0.01) in ambient temperatures <20 degrees C (35.9+/-1.4 degrees C, n=225) compared to > or =20 degrees C (36.5+/-0.9 degrees C, n=1450). Among hypothermic newborns, 42% (331/787) of their mothers had a lower temperature (range -6.7 to 0.1 degrees C, mean difference 0.4+/-1.2 degrees C). Acceptance of STSC was nearly universal. No adverse events from STSC were reported. STSC was perceived to prevent newborn hypothermia, enhance mother's capability to protect her baby from evil spirits, and make the baby more content. CONCLUSION: STSC was highly acceptable in rural India when introduced through appropriate cultural paradigms. STSC may be of benefit for all newborns and for many mothers as well. New approaches are needed for introduction of STSC in the community compared to the hospital.

Phacotrabeculectomy Versus Conventional Combined Technique in Coexisting Glaucoma and Cataract.

BACKGROUND: To evaluate and compare efficacy and outcome after single site phacotrabeculectomy and conventional combined surgery in cases of coexisting primary open angle glaucoma and cataract. METHODS: This prospective study on fifty patients of concurrent primary open angle glaucoma and cataract, who had undergone combined surgery as single site phacotrabeculectomy or conventional single site trabeculectomy with extracapsular lens extraction with IOL implantation in 25 cases each. Evaluation was based on operative and postoperative complications, control of IOP and visual outcome. The follow up period ranged between twelve months to eighteen months. RESULTS: The mean medically controlled preoperative intraocular pressure was 22 mm of Hg (Range 18 to 35 mm of Hg) by applanation method of tonometry. The range of postoperative intra-ocular pressure after one year was 11 to 22 mm of Hg in first and 14 to 26 mm Hg in second group. Failure to maintain optimum postoperative IOP without Beta-blocker was more frequent after conventional combined procedure. There was no significant difference in incidence and pattern of postoperative complications. CONCLUSION: Phacotrabeculectomy provides effective and sustained visual recovery and adequate control of intraocular pressure as compare to conventional combined procedure.

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor.

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.

Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor.

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac alpha-actin, skeletal alpha-actin, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation.

BACKGROUND: Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. RESULTS: Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the alpha-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. CONCLUSION: Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This facilitates the comparative analysis of lethal alleles and thereby advances our ability to analyze gene function in mammals.

Calcium and disease: molecular determinants of calcium crystal deposition diseases.

Deposition of basic calcium phosphate (hydroxyapatite, octacalcium phosphate and tricalcium phosphate) (BCP) and crystalline calcium pyrophosphate dihydrate (CPPD) is associated with a variety of aging-related pathologies, including osteoarthritis, cartilage degeneration and pseudogout. These diseases of calcium deposition serve as some of the best-studied examples of how calcium-regulated changes in gene expression can directly lead to pathogenic consequences. Tissue damage can result when crystals stimulate cells to release matrix-degrading molecules or secrete cytokines that stimulate the release of matrix-degrading molecules. Exposure of cultured cells to crystals induces expression of cellular proto-oncogenes such as c-fos, c-myc and c-jun, by a calcium-dependent mechanism, and this response can be blocked by a potential therapeutic compound, phosphocitrate. Activation of the c-fos and c-jun genes is directly involved in expression of metalloproteinases such as collagenase and stromelysin, suggesting that crystal-mediated activation of these genes is directly involved in pathogenesis. In this review recent advances in the molecular mechanisms responsible for crystal-mediated cell activation are discussed.

Serum response factor-dependent regulation of the smooth muscle calponin gene.

Smooth muscle calponin is a multifunctional, thin filament-associated protein whose expression is restricted to smooth muscle cell lineages in developing and postnatal tissues. Although the physiology of smooth muscle calponin has been studied extensively, the cis-elements governing its restricted pattern of expression have yet to be identified. Here we report on smooth muscle-specific enhancer activity within the first intron of smooth muscle calponin. Sequence analysis revealed a proximal consensus intronic CArG box and two distal intronic CArG-like elements, each of which bound recombinant serum response factor (SRF) as well as immunoreactive SRF from smooth muscle nuclear extracts. Site-directed mutagenesis studies suggested that the consensus CArG box mediates much of the intronic enhancer activity; mutating all three CArG elements abolished the ability of SRF to confer enhancer activity on the smooth muscle calponin promoter. Cotransfecting a dominant-negative SRF construct attenuated smooth muscle-specific enhancer activity, and transducing smooth muscle cells with adenovirus harboring the dominant-negative SRF construct selectively reduced steady-state expression of endogenous smooth muscle calponin. These results demonstrate an important role for intronic CArG boxes and the SRF protein in the transcriptional control of smooth muscle calponin in vitro.

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals.

Serum Response Factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth, differentiation, and development. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied, however, less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including an ETS domain binding site, an overlapping Sp1/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces the SRF gene by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation by cells by lysophosphatidic acid (LPA) or whole serum, the SRF promoter is upregulated by a bipartite pathway that requires both an Sp1 factor binding site and the CArG motifs for maximal stimulation. The CArG box-dependent component of this pathway is targeted by Rho mediated signals, and the Sp1 binding site dependent component is targeted by Ras mediated signals.

Basic fibroblast growth factor activates serum response factor gene expression by multiple distinct signaling mechanisms.

Serum response factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth and differentiation. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied; however, significantly less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including a consensus ETS domain binding site, a consensus overlapping Sp/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces SRF by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation of cells by a purified growth factor, basic fibroblast growth factor (bFGF), the SRF promoter is upregulated by a complex pathway that involves at least two independent mechanisms: a CArG box-independent mechanism that is mediated by an ETS binding site, and a novel CArG box-dependent mechanism that requires both an Sp factor binding site and the CArG motifs for maximal stimulation. Our analysis indicates that the CArG/Sp element activation mechanism is mediated by distinct signaling pathways. The CArG box-dependent component is targeted by a Rho-mediated pathway, and the Sp binding site-dependent component is targeted by a Ras-mediated pathway. Both SRF and bFGF have been implicated in playing an important role in mediating cardiogenesis during development. The implications of our findings for SRF expression during development are discussed.

Conjunctival melanoma in the black population.

Melanoma of the conjunctiva is a rare, unilateral malignancy primarily affecting middle-aged whites; the annual average age-adjusted incidence rate is 0.012 per 100,000 population. Although conjunctival melanoma in the black population is extremely rare, cases have been reported. Melanoma of skin in blacks has a predilection for nonsun-exposed, nonpigmented sites such as mucous membranes, palms, and soles. Primary acquired melanosis may lead to the development of melanoma even in blacks. Primary acquired melanosis in the black population may be difficult to differentiate from racial melanosis clinically and histopathologically. Early diagnosis through awareness and education can help improve the survival of black patients with conjunctival melanoma.

Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites.

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a stoichiometry of approximately 2 molecules of ADP-ribose incorporated per molecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at more than one arginine residue. SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility than non-ADP-ribosylated Ras. Analysis of the ADP-ribosylation of in vitro transcribed/translated Ras by ExoS identified two electrophoretically shifted forms of Ras, which was consistent with the ADP-ribosylation of Ras at two distinct arginine residues. Analysis of ADP-ribosylated in vitro transcribed/translated Ras mutants possessing individual Arg-to-Ala substitutions showed that Arg-41 was the preferred site of ADP-ribosylation and that the second ADP-ribosylation event occurred at a slower rate than the ADP-ribosylation at Arg-41, but did not occur at a specific arginine residue. Analysis of bacterially expressed wild-type RasDeltaCAAX and RasDeltaCAAXR41K supported the conclusion that Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is located adjacent to the switch 1 region of Ras, which is involved in effector interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited nerve growth factor-stimulated neurite formation. This is the first demonstration that ExoS disrupts a Ras-mediated signal transduction pathway.

Phosphocitrate inhibits a basic calcium phosphate and calcium pyrophosphate dihydrate crystal-induced mitogen-activated protein kinase cascade signal transduction pathway.

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10(-3) to 10(-5) M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10(-3) M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP- and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1beta induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.

Expression of the serum response factor gene is regulated by serum response factor binding sites.

The serum response factor (SRF) is a ubiquitous transcription factor that plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. Notably, SRF has been found to be a key regulator of members of a class of cellular response genes termed immediate-early genes (IEGs), many of which are believed to be involved in regulating cell growth and differentiation. The mechanism by which SRF activates transcription of IEGs in response to mitogenic agents has been extensively studied. Significantly less is known about how expression of the SRF gene itself is mediated. We and others have previously shown that the SRF gene is itself transiently induced by a variety of mitogenic agents and belongs to a class of "delayed" early response genes. We have cloned the SRF promoter and in the present study have analyzed the upstream regulatory sequences involved in mediating serum responsiveness of the SRF gene. Our analysis indicates that inducible SRF expression requires both SRF binding sites located within the first 63 nucleotides upstream from the start site of transcriptional initiation and an Sp1 site located 83 nucleotides upstream from the start site. Maximal transcriptional activity of the promoter also requires two CCAATT box sites located 90 and 123 nucleotides upstream of the start site.

Observer variation in AgNOR counts in neoplastic breast lesions.

OBJECTIVE: To determine interobserver and intraobserver variability of AgNOR quantitation in neoplastic lesions of the breast. STUDY DESIGN: Forty-five cases, 20 benign and 25 malignant lesions, were included in the study. Counts were performed on one slide from each case within a pre-marked area of about 1 cm2 in a standardized manner by two observers blind to the histopathologic diagnosis and independent of each other and repeated after two weeks. Interobserver and intraobserver agreement was assessed using the Bland-Altman method. RESULTS: Our results showed small mean interobserver and intraobserver differences but wide limits of agreement. CONCLUSION: Observer variation in AgNOR counts is too high for the method to be of any diagnostic or prognostic relevance.

NESTED CASE - CONTROL ANALYSIS OF THE RISK FACTORS FOR HIGH ALTITUDE PULMONARY OEDEMA.

A nested case-control study was undertaken on a cohort of soldiers inducted into high altitude area (11000 to 16000 feet) of Western Himalayas, with the objectives of studying the incidence of high altitude pulmonary oedema (HAPO) and its association with physical exertion and certain other predetermined risk factors. The study indicated that the cumulative incidence of HAPO was 1.42 per 1000 inductions. The association with moderate/strenuous physical exertion within 24 hours of entry into high altitude was significant (Odds ratio (OR) = 3.19; 95% confidence limits (CL) = 1.23 to 8.15); however, this association was not significant for the period 24 to 48 hours or > 48 hours. Physical exertion during first 24 hours was also significantly associated with severity of disease (OR = 14.67, 95% CL = 3.61 to 64.04), but not after 24 hours. Previous history of "high altitude sickness" was also significantly associated with HAPO (OR = 2.74, 95% CL = 1.12 to 6.77). Physical exertion during first 24 hours was found to carry an attributable risk of 2.56 per 1000 inductions and an etiologic fraction of 17.8%. No significant association of HAPO was observed with age, type of inductee (fresh/reinductee), native place, alcohol consumption and smoking.

Capnocytophaga keratitis. A clinicopathologic study of three patients, including electron microscopic observations.

BACKGROUND: Histopathologic studies of this unusual keratitis caused by Capnocytophaga species have not been reported previously. METHODS: The authors report the light microscopic and ultrastructural findings of three patients with a distinctive necrotizing keratitis caused by an anaerobic gram-negative bacillus. In three patients, ages 19, 81, and 91 years, a necrotizing stromal keratitis developed; two of these patients had a previous penetrating keratoplasty for pseudophakic bullous keratopathy. The first patient did not have ocular surgery previously and was treated initially for presumed Acanthamoeba keratitis. RESULTS: By light microscopy, all three keratectomy specimens were strikingly similar and showed a necrotizing and/or suppurative stromal keratitis displaying myriad slender, fusiform, gram-negative bacilli located anterior to Descemet's membrane and extending into the deep corneal stroma, assuming a "picket fence" appearance. Cultures of the cornea in case 1 grew Capnocytophaga ochracea. For the remaining two patients, a diagnosis presumptively was made based on characteristic histopathologic features. Results of electron microscopic examination showed numerous bacilli that were mostly extracellular; occasional organisms were phagocytosed by macrophages. CONCLUSION: The histopathologic features of Capnocytophaga keratitis are unique; therefore, a presumptive diagnosis can be made based on the morphology and location of the bacilli in the keratectomy specimens. To the authors' knowledge, this is the first study describing the typical histopathologic and electron microscopic findings of Capnocytophaga keratitis.

L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway.

A mechanism by which calcium-induced signals are transduced to the nucleus to activate transcription of the c-fos proto-oncogene has been characterized. The serum response element (SRE), a region of the c-fos gene which controls growth factor-induced transcription, is now shown to mediate c-fos transcription in response to activation of L-type voltage-sensitive calcium channels. Calcium-dependent transcriptional activation through the SRE is mediated by the serum response factor (SRF). Membrane depolarization induces phosphorylation of SRF at Ser-103, an event shown to enhance the ability of SRF to bind the SRE. Ca(2+)-induced SRF phosphorylation occurs via a pathway that may involve Ca2+/calmodulin-dependent kinases.

CLINICO-EPIDEMIOLOGICAL ALGORITHM FOR PREDICTING SYSTEMIC ARTERIAL HYPERTENSION AT HIGH ALTITUDE THROUGH MATHEMATICAL MODELLING.

A population based hybrid design combining element of cohort and cross-sectional approach was used to develop a simple clinical algorithm to predict individual probability of developing hypertension (systolic BP > 140 mm Hg and/or diastolic BP > 90 mmHg). 3615 soldiers initially normotensive at the time of induction into high altitude, were studied by systematic random sampling. Multiple logistic regression analysis showed a high significant association between hypertension and age, body mass index (BMI), tobacco smoking and alcohol consumption. Using the constant/coefficient values obtained from the logistic model and the receiver operating characteristics (ROC) curve analysis, the following predictive rule was developed - To the age in years, add (BMIx 3.86); also add 5.53 if he is a smoker; and add 19.81 if he consumes alcohol. If the total exceeds 142, the individual is at high risk of developing hypertension. This algorithm carries a sensitivity of 68.2% and specificity of 78.5%.