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.

The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts.

Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.

A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

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.

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.

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.

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.

Renal gamma-glutamyltranspeptidase: In situ antiluminal localization.

Renal localization of gamma-glutamyltranspeptidase was studied in functioning kidneys perfused with the gamma-glutamyl substrates, gamma-glutamyl-p-nitroanilide and N(gamma-L-glutamyl) 2-naphthylamide. In kidneys perfused with gamma-glutamyl-p-nitroanilide, 90% of the breakdown products appear in the blood (perfusate) rather than urine. The addition of the acceptor substrate glycylglycine to the perfusate stimulated gamma-glutamyl-p-nitroanilide breakdown 3-fold, both on the blood and urine sides, while the appearance of gamma-glutamylglycylglycine confirmed that the activity responsible stems indeed from gamma-glutamyltranspeptidase. Ureter ligation reduced the glomerular filtration rate to less than 10% of the control, while the breakdown of gamma-glutamyl-p-nitroanilide decreased only 5%. Kidneys perfused with N(gamma-L-glutamyl) 2-naphthylamide and the diazonium salt demonstrated staining of the glomeruli; isolated glomeruli and microvessels gave heavy staining when incubated with the histochemical substrates. These results demonstrate the association of a significant portion of the total renal gamma-glutamyltranspeptidase activity with the microvascular compartment.

A partial-thickness epithelial defect increases the adherence of Pseudomonas aeruginosa to the cornea.

Some patients with infectious keratitis have no clinically demonstrable corneal abrasion predisposing them to infection. Subtle, undetectable corneal injuries may facilitate bacterial adherence to the cornea, eventually leading to keratitis. To study this concept, we have developed a rabbit model in which a partial-thickness corneal epithelial defect was induced by filter paper impression on the cornea that removed one to two layers of corneal epithelium. Following this injury, the corneas were incubated with Pseudomonas aeruginosa, washed, and the number of bacteria adhering to the injured corneas as well as to control corneas was quantitated. Corneas treated with filter paper, either ex vivo or in vivo, allowed 20 times more bacteria to adhere than did the untreated control corneas (P less than 0.01). This superficial epithelial defect increased Pseudomonas adherence to the cornea for up to 72 hr after injury. When corneal injury was extended to the stroma, the adherence of Pseudomonas was further augmented as compared to adherence to the superficially injured cornea. Thus, we conclude that a clinically subtle, partial-thickness corneal epithelial injury can markedly facilitate the adherence of Pseudomonas aeruginosa, which may be an important predisposing factor for infectious keratitis.

Multiple pterygium syndrome.

We describe a sporadic case and four sibs from a consanguineous Nicaraguan family affected with the multiple pterygium syndrome. Clinical manifestations included normal intelligence; short stature; pterygia of neck, axillary, antecubital, popliteal, digital, and intercrural areas; multiple joint contractures with a crouched stance; a flat, sad, motionless facial appearance; and cleft palate. Males had small penis and scrotum and cryptorchidism; females had apparent aplasia of labia majora and small clitoris. Skeletal anomalies included fusion of cervical vertebrae, scoliosis, flexion contractures of fingers and "rocker-bottom" feet with vertical talus. This review documents genetic heterogeneity: Autosomal recessive inheritance in many cases, autosomal dominant determination in others, and sporadic occurrence.

Carbohydrate deposits on the surfaces of worn extended-wear soft contact lenses.

Three different commercial extended-wear soft contact lenses worn continuously by patients for at least 28 days were stained with fluorescein isothiocyanate-labeled lectins. These lectins detected the presence of alpha-linked or beta-linked D-mannose, D-glucose, D-galactose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetyl neuraminic acid (sialic acid) on the surfaces of the contact lenses. These saccharides are bound to other sugars that likely account for an integral part of glycoprotein and/or glycolipid deposits on lens surfaces. These tear deposits may contribute to the chemical spoilage of the lens and, furthermore, may serve as specific receptors for pathogenic microorganisms commonly implicated in extended-wear soft contact lens-associated infectious keratitis.

Use of carbon fibers for repair of abdominal-wall defects in rats.

Carbon in the form of 8-micron fibers induces growth of connective tissue. The purpose of this study was to measure and histologically characterize tissue ingrowth occurring in carbon fibers implanted for up to 12 months in abdominal-wall defects in rats, compared with polypropylene mesh. Carbon fibers induced significantly more tissue ingrowth than polypropylene mesh at 6 to 12 months postoperatively. The predominant tissues associated with carbon fibers and polypropylene mesh were dense connective tissue and fat, respectively. Fragmentation of the implants did not occur, and implant debris was not found in the regional lymph nodes. Carbon fibers are potentially useful for reinforcing abdominal-wall defects.

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.

Metastasizing basal cell carcinoma originating in a stasis ulcer in a black woman.

A case of basal cell carcinoma originated in a long-standing stasis ulcer on the lower extremity and then metastasized to the dermis and subcutaneous tissue of the thigh. The literature on metastasizing basal cell carcinoma is reviewed.

Contact lens wear enhances adherence of Pseudomonas aeruginosa and binding of lectins to the cornea.

Extended wear soft contact lenses are associated with an increased incidence of Pseudomonas aeruginosa keratitis. Because the first step in the pathogenesis of this disease is adherence of the microorganism to the corneal surface, we studied the effect of soft contact lens wear on the adherence of P. aeruginosa to the cornea. Rabbits were fitted for extended wear soft contact lenses in the left eye, and the right eye served as a control. Both eyes were then closed with a partial tarsorrhaphy. After 1-5 days of wear, the lenses were removed and the corneas of the left and right eye were removed. Differences in the number of adherent Pseudomonas and in lectin binding to lens-wearing corneas and non-lens-wearing corneas were determined. After 1, 3, and 5 days of soft contact lens wear, there was a significant increase in the number of P. aeruginosa adherent to the lens-wearing cornea. Three to eight times as many bacteria adhered to the lens-wearing eye as compared with the control eye (p less than 0.05). In addition, a soft contact lens placed in the eye followed by the immediate application of P. aeruginosa resulted in an eightfold increase in adherence of bacteria to the lens-wearing cornea (p less than 0.05). Lens wear also led to an increase in binding of concanavalin A (Con A), wheat germ agglutinin (WGA), and Maclura pomifera agglutinin (MPA) to surface epithelium covered by the lens. These corneal epithelial changes induced by extended wear soft contact lenses may provide some insight as to why soft contact lens wearers are predisposed to Pseudomonas keratitis.

The contribution of bacterial surface hydrophobicity to the process of adherence of Pseudomonas aeruginosa to hydrophilic contact lenses.

Ten isolates of Pseudomonas aeruginosa obtained from the corneas of patients with Pseudomonas keratitis adhered to soft contact lenses in significantly greater numbers than did six isolates from other body sites (P less than .05). However, there was no predominant serotype among the 10 corneal isolates tested. Isolates grown statically in broth at 37 degrees C formed a pellicle and adhered two times as much to contact lenses as did isolates grown in broth while shaking which did not form a pellicle (P less than .01). The more adherent isolates (grown at 37 degrees C) were shown to be more hydrophobic than the less adherent bacteria (grown at 26 degrees C) by their propensity to accumulate at the interface between hexadecane and saline and their movement into polyethylene glycol from dextran. These corneal isolates agglutinated erythrocytes, a process that was inhibited by dilute solutions (as low as 0.01%) of three commonly used surfactants. These same surfactants inhibited the adherence of Pseudomonas aeruginosa to soft contact lens surfaces by as much as 52%. It is concluded that hydrophobic interactions may significantly contribute to the ability of Pseudomonas aeruginosa to adhere to contact lenses.

Isolation of a herpes simplex virus type 2 that is retinovirulent in mice.

The virulence of a herpes simplex virus type 2 (HSV-2) isolated from the urine of a patient (SL) with acquired immunodeficiency syndrome (AIDS) and bilateral acute retinal necrosis (ARN), was investigated in mice. The ratio of plaque forming units (PFU) in fibroblasts to the 50% lethal dose (LD50) of HSV-2(SL) in mice was 10 fold more than the PFU to LD50 ratio of a neurovirulent HSV-2, strain 186. Further, HSV-2(SL) caused retinitis with and without lethal encephalitis in mice inoculated intracranially (i.c.). In contrast, mice inoculated with HSV-2(186) died of encephalitis without ocular disease. HSV-2(SL) was isolated from eye and/or brain tissue 1 to 15 days post i.c. inoculation. Ocular disease progressed from an initial mild chorioretinitis on day 8 to total retinal necrosis with panuveitis by day 11 in mice given 10 PFU of HSV-2(SL) i.c. HSV antigen was detected initially in the cells of the optic nerve and spread into the ganglial cells of the nerve fiber layer, the neurosensory cells of the inner nuclear layer, and the cells of the retinal pigment epithelium (RPE) between days 8 and 10. Thus, this study supports the concept that HSV neurovirulence varies between strains and presents a HSV-2 neurotransmission animal model of ARN.

Recent studies on 'hyalinized' glomeruli.

Hyalinized glomeruli in tissue sections those isolated from kidneys of subjected with end-stage and diabetic nephropathies and from aged persons (older than 75 years) were studied by histochemical, immunohistochemical, electron microscopic, and chemical methods. Our purposes were as follows: (1) to clarify the pathogenic mechanisms of glomerular hyalinization; (2) to resolve some of the controversies on terminology in the literature. Results showed significant increases in the amounts of collagen (13% to 30%), carbohydrates (30% to 47%), and lipids (58% to 82%), and decreases in the amounts of DNA (40%) and RNA (40% to 50%) in the hyalinized glomeruli of diseases compared with normally sized matched glomeruli. I conclude that glomerular hyalinization is a complex molecular pathogenic process.

Spondyloepiphyseal dysplasia congenita. A comparative study of chondrocytic inclusions.

Spondyloepiphyseal dysplasia congenita is a short-trunk chondrodysplasia trait. The abnormalities are present at birth and involve primarily the spine, the epiphyses of long bones, and the pelvis. Postmortem examinations of two patients with this disease who died shortly after birth showed a mild disorganization of chondrocytic columnization in the physeal growth zone. The chondrocytes contained PAS-positive cytoplasmic inclusions after diastase digestion to eliminate glycogen. Ultrastructural examination of the inclusions in one patient showed them to be accumulations of finely granular material in dilated cisterns of rough endoplasmic reticulum. To our knowledge, similar findings have been seen only in three other types of chondrodysplasia. The inclusions, which therefore are of diagnostic importance, may be of importance also in reflecting the primary metabolic abnormality.

Blastomycotic meningitis.

A difficult and tragic case of central nervous system blastomycosis is presented as the basis for a review of the diagnostic criteria required to establish this diagnosis and to present a scheme for the diagnosis and therapy of chronic meningitis. The diagnosis in our case was complicated by preexisting inadequately treated tuberculosis; a prepontine mass; and a cervical intradural, extramedullary, circumferential mass. This exceptional case of chronic basilar meningitis with cervical myelopathy was caused by Blastomyces dermatitidis.