Activating mutation of the Ha-ras gene in chemically induced tumors of the hamster cheek pouch.

The presence of an activating mutation in the Ha-ras gene in hamster cheek pouch tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) complete carcinogenesis was investigated. The normal sequence of a fragment of genomic DNA encompassing codon 61 of the Ha-ras gene was amplified by the polymerase chain reaction using primers designed for a highly conserved region of the mouse Ha-ras-1 gene. The sequence of the amplified fragment was determined by a direct sequencing technique and exhibited 83.3% and 87.5% homology with the corresponding human and mouse sequences, respectively. At the amino acid level, the sequence was identical among the three species. Paraffin sections of 11 squamous cell carcinomas of the cheek pouch were used to detect mutated Ha-ras alleles. DNA sequencing of the tumors showed that six of 11 tumors presented an A----T transversion in the second position of codon 61, resulting in an amino acid change from glycine to leucine. As has been demonstrated in other systems, we have shown a specific mutation of the Ha-ras gene in chemically induced tumors of the hamster cheek pouch, further supporting the role of this oncogene in chemical carcinogenesis.


Evidence for coincident mutations in human lymphoblast clones selected for functional loss of a thymidine kinase gene.

A mitotic "recombination-competent state" inducible by x-irradiation is thought to exist in yeast. We sought evidence for such a process in mammalian cells by examining the occurrence of mutations at unlinked loci in clones derived from a human lymphoblast cell line. A total of 169 independent clones that arose spontaneously or after exposure to x-rays or ethyl methanesulfonate were selected for new somatic mutations at the thymidine kinase gene on chromosome 17q. They were subsequently screened for coincident mutations by use of variable-number-of-tandem-repeat probes located on different chromosomes. Three coincident mutations were positively identified by Southern analysis on chromosomes 7 and 14; they included one that produced a new allele and two that caused loss of allele heterozygosity. Densitometric analysis of the latter two indicated the presence of two copies of the remaining allele. Several possible coincident genetic events were also observed on chromosome 17. These findings revealed a coincident mutant fraction of about 10(-2)/cell, whereas the expected mutation fraction at these loci is less than 10(-4)/cell. These results may thus provide the first molecular evidence that a "global" mutational process capable of inducing genetic instability exists in mammalian cells.


Differential retention of tumor- and differentiation-suppressor functions in cells derived from a human squamous cell carcinoma.

Three morphologically distinct cell lines--F.2a, V, and B.2--were isolated from a single human squamous cell carcinoma. Although all three cell lines can grow indefinitely in culture, they differ in a number of important transformation-related phenotypes. Only B.2 is strongly tumorigenic when injected into the flanks of nude mice, and only V can efficiently grow in semisolid media. The dominance of these traits was investigated by generating somatic cell hybrids among the three cell lines. F.2a was able to suppress the tumorigenicity of B.2 cells, whereas B.2 inhibited the capacity for anchorage-independent growth of V, the latter trait being a function of the ability of these epithelial cells to differentiate when deprived of support. The influence of exogenously added growth factors was also evaluated. This study indicates that the particular tumor we examined consisted of a heterogeneous population of cells with distinct growth and differentiation capacities.


Approaches to patient and family education for pressure ulcer management.

The Agency for Health Care Policy and Research (AHCPR) guidelines for pain management, urinary incontinence, and pressure ulcers emphasize the importance of patient and family education. Patient teaching strategies are discussed by using the example of pressure ulcer management. The family is invited into an educational partnership through needs assessment and mutual goal setting. Teaching strategies that involve the learner and are adapted for the home setting are most effective. Evaluation of teaching should be in terms of patient outcomes.


Photographing pressure ulcers to enhance documentation.

Complete and careful observation, description and documentation of pressure ulcers is an essential component of practice when caring for people with pressure ulcers. Photographing pressure ulcers and including the photo as part of the patient's chart provides a useful adjunct to the written record. While a "picture is worth a thousand words," photographs can be misleading unless they are consistently taken according to specific guidelines. Techniques to enhance photographic results and avoid common errors are discussed.


Pressure ulcer risk assessment--simple or complex?

A descriptive comparison study was carried out to determine whether there was a difference between a staff nurse's single-item pressure ulcer risk assessment and a risk assessment using the Braden Scale. Seventy-two medical patients participated in the study. Statistical analysis of the data indicated nurses were accurate in predicting pressure ulcer risk with a simple YES/NO answer. The mean Braden Score for those patients indicated at risk by the nurse was 14.45, and 20.24 for those assessed to be not at risk. This study indicates that nurses' good judgement is as reliable as more complicated tools in determining pressure ulcer risk. Further studies comparing risk assessment tools are recommended.


Teaching the assessment of patients with pressure ulcers.

Teaching assessment of patients with pressure ulcers involves the teaching of assessment of the total person as well as at-risk potential, daily examination of skin, and accurate assessment of progress or regression of a pressure ulcer. Suggestions and references are provided for teaching each of these aspects of assessment.


Liposomes in the treatment of malignancy: a clinical perspective.

Technological advances in liposomal preparation and efficient drug entrapment, along with supportive preclinical studies, have led to a number of recent clinical trials utilizing liposomes as drug carriers in the treatment of human malignancy. Although the results of these trials must be considered preliminary, it is clear that liposomal delivery of chemotherapeutic agents is safe at the doses administered. Aside from minor constitutional symptoms, virtually all toxicity could be attributed to release of the incorporated drug. Myelosuppression tends to be the dose-limiting toxicity with free drug, whereas constitutional symptoms are more likely to occur with encapsulated biologic therapy. Prior to human trials, there was fear that intravenous injection of liposomes could result in pulmonary emboli. No cases of pulmonary embolism secondary to liposome therapy have been recorded. The objective response rate in the patients studied appears to be minimal. This is not surprising, since the overwhelming majority of patients studied had disease that was advanced and previously shown to be refractory to therapy. Subgroups of patients that appear to benefit most include those with breast cancer who were treated with liposomal doxorubicin and those with advanced melanoma treated with liposomal tumor vaccines. Additional phase II and III clinical trials will better define the effectiveness of treatment modalities incorporating liposomes. VI-A. Future directions One of the earliest applications of liposomes may be in the amelioration of drug toxicity. Although not yet proven, the clinical studies reviewed suggest that liposomal delivery of doxorubicin reduces cardiotoxicity without sacrificing antitumor effect. Although similar claims have been made in support of continuous infusion doxorubicin [11], one can avoid unnecessary hospitalization or the bulk and expense of portable infusion devices by a single administration of the liposomal preparation. Liposome encapsulation can markedly alter the biodistribution and pharmacokinetics of well-known chemotherapeutic agents. The effectiveness of liposomal drug delivery in human trials thus far has probably been more closely related to altered pharmacokinetics rather than enhanced drug delivery to tumor or increased tumor responsiveness. As demonstrated by Gabizon [19], increased liposome circulating time in the murine model can be achieved by using small unilamellar vesicles containing a phosphatidylcholine of high phase-transition temperature and a small molar fraction of monosialoganglioside or hydrogenated phosphatidylinositol.(ABSTRACT TRUNCATED AT 400 WORDS)


Nutritional support of the child with cancer.

The purpose of this article is to outline how the disease, treatment, and psychological state of a child with cancer impact on the child's nutritional status. The methods of assessing nutritional status, including anthropometric measurements, laboratory indices, clinical observation, dietary assessment, and psychosocial evaluation, are summarized. After nutrition assessment, the pediatric oncology nurse and the dietitian, along with the oncologist, the family, and the child, develop a plan of care. The specific roles of the pediatric oncology nurse and dietitian in the nutrition intervention are described.


Pain in the pediatric oncology patient.

Pediatric oncology nurses face many challenges in treating the pain associated with childhood cancer. The type and severity of pain children with cancer experience varies from acute, short-term, procedure-related pain to the progressive chronic pain associated with terminal illness. In addition, the unfounded fears of using strong narcotic analgesics and the underutilization of psychological techniques to treat pain in children limit the effectiveness of pain management. Armed with objective data, pediatric oncology nurses can work with other members of the cancer treatment team to provide relief from the pain associated with the diagnosis and treatment of childhood cancer.


Nursing research--moving forward through networking, collaboration, and mentorship.

Numerous pediatric oncology nurses have provided the specialty with research that has established a foundation for the nursing care of children and adolescents with cancer. The research committee within APON is committed to facilitating further research development among pediatric oncology nurses. Research growth within APON will continue with the development of precongress research workshops for nurses interested in developing research projects, an advanced nursing research session to be held at the next national conference, and the organization of networking among pediatric oncology nurse researchers. Additionally, the research column will provide nurses with a review of the research process over the next several issues. Knowledge building is an evolving, dynamic process that is rapidly changing due to the newness of the specialty. As the pediatric oncology specialty continues to grow, networking, collaboration, and mentorship must become the keys to facilitating future research growth within APON.


Gastrointestinal manifestations in the child with cancer.

Pediatric oncology nurses provide a major role in the assessment and management of gastrointestinal complications in children resulting from cancer therapies. The clinical problems most frequently seen in this area include stomatitis, nausea, vomiting, constipation, and diarrhea. These areas are reviewed in detail according to various nursing diagnoses including definitions and pathophysiology, recent studies and interventions, special considerations for children, and patient and parent education.


Minerals and trace elements in milk.

The nutritional roles, requirements, and metabolism and the quantitative relationship between dietary intakes and health for a number of the minerals and trace elements have been more clearly defined in recent years, but there are still considerable deficiencies in our understanding of these issues, e.g., the significance of calcium in the etiology and treatment of osteoporosis and hypertension. Reliable information is now available on the content, and the principal factors affecting it, of most of the minerals and trace elements in human and cow's milks. However, for some of the trace elements, there is still a wide variation in reported values in the literature, which is due, at least in part, to analytical difficulties. The contribution of cow milk and milk products to the diet in Western countries is significant for sodium, potassium, chloride, calcium, phosphorus, zinc, and iodine. Iodine is the only trace element for which there has been any suggestion of excessive amounts in cow milk. However, there is evidence of a decline in milk iodine concentrations in the United States in recent years, although the situation in other countries less clear. Breast milk usually has adequate mineral and trace element contents for feeding full-term infants, with the exceptions of fluoride, for which supplementation of infants is recommended, and of selenium in some countries, such as Finland and New Zealand, where maternal intakes are low. However, breast milk selenium contents have increased in these countries in recent years due to increased maternal selenium intakes. The concentrations of minerals and trace elements in infant formulas for full-term infants are generally higher than in human milk, and all appear to be more than adequate, with the possible exception of selenium, which may need to be increased in some formulas. Considerable changes in the mineral and trace element contents of formulas have been instituted in recent years in the light of improved knowledge of infant requirements. While the chemical forms of the macrominerals and some of the trace elements (iron, zinc, copper, and manganese) in milks are fairly well defined, the forms of many of the trace elements are unknown. Sodium, potassium, chloride, and iodine are believed to be almost totally absorbed from milks and infant formulas.(ABSTRACT TRUNCATED AT 400 WORDS)


Dietary fatty acids, lipoproteins, and cardiovascular disease.

Dietary fat quality and quantity significantly affect the metabolism of all the plasma lipoproteins and probably constitute the most significant dietary determinants of plasma lipoprotein levels. Since the major role of the plasma lipoproteins is the transport of exogenous and endogenous fat, this would be expected of a highly regulated, metabolically homeostatic system. The data clearly show that dietary fat saturation affects all aspects of lipoprotein metabolism, from synthesis to intravascular remodeling and exchanges to receptor-mediated and nonspecific catabolism. The experimental data regarding dietary fatty acid effects on lipoprotein metabolism are complicated and at times contradictory due to the large degree of metabolic heterogeneity in the population, which, when coupled with the known abnormalities of lipoprotein metabolism associated with certain types of hyperlipoproteinemia, can present responses from A to Z. It is clear that the same dietary pattern has different effects in different individuals and that complicating factors of individuality raise some concerns regarding generalized dietary recommendations. As new knowledge of the role of dietary factors and CVD risk develops, and our abilities to characterize the individual patient's response to dietary interventions become more refined, it may be possible to specify dietary fat intervention from a patient-oriented concept rather than a single all-purpose diet approach. Thus it would be possible to design dietary interventions to match patient needs and gain both efficacy and compliance. With the spectrum of approaches possible--low fat, moderate fat with MUFA, n-3 PUFA, etc.--we should be able to approach dietary interventions to reduce CVD risk at both a population-based level and a patient-specific level. There remains much to learn regarding the effects of dietary fatty acids on the synthesis, intravascular modifications, and eventual catabolism of the plasma lipoproteins. The area of lipoprotein metabolism in health and disease, of its modifications by diets and drugs, and of the contributions of genetic heterogeneity to these processes is one of notable advances over the past two decades and continues to be an area of intense investigation.


Metabolism of drugs: a reappraisal.

The paper deals with an up-to-date reappraisal of the most relevant aspects connected to the investigation of drug metabolism including the most appropriate methods used in preclinical and clinical field. Renal and biotransformation clearance is considered with several specific references. Reversible metabolism, active metabolites of drugs, various kinds of first pass effect, genetic polymorphism in drug metabolizing enzymes, the most appropriate pharmacokinetic analysis and regulatory aspects are the main aspects focused on.


Origin of the two mRNA species for the human neurofilament light gene.

The human neurofilament light (hNF-L) gene yields two major species of mRNAs of 2.4 and 3.8 kilobases (kb) in size. To investigate the origin of these two mRNAs, we have analyzed in transgenic mice the expression of hNF-L DNA fragments including different lengths of 5'-flanking regions. The finding that the 3.8-kb mRNA species is produced by a hNF-L transgene that includes only the proximal promoter region (-0.3 kb) demonstrates that both the 2.4- and 3.8-kb mRNAs are derived from the same site of transcription initiation. Sequencing of the 3' untranslated region of the hNF-L gene revealed the presence of multiple AATAAA polyadenylation signals. We conclude from Northern blotting experiments using probes spanning various regions of the hNF-L gene that the 2.4- and 3.8-kb mRNAs originate from the selective use of polyadenylation signals located 1.4 kb apart.


Identification of proteins potentially involved in proximal-distal pattern formation in the regenerating forelimb of the newt.

We have used high resolution two-dimensional gel electrophoresis to identify and characterize proteins that may represent products of genes involved in establishing positional information along the proximal-distal axis of the regenerating forelimb of the newt Notophthalmus viridescens. At least 24 proteins have been found whose synthesis and (or) abundance is increased in proximal (midstylopodial) regenerates relative to midzeugopodial (distal) regenerates at either of two regeneration stages, the early dedifferentiation and moderate bud stages. Four of these same proteins show an axial asymmetry at both stages. Ten distal-specific proteins were also identified, although only one was common to both stages. More significantly, 6 of these 34 proteins (molecular masses of 73, 73, 51.5, 44.0, 19.5, and 16.5 kilodaltons and isoelectric points of 6.70, 6.74, 6.0, 6.05, 5.9, and 6.98, respectively) are regulated to proximal levels by treatment of distal regenerates with retinoic acid (RA) at both stages. An additional five are proximalized by RA at only one regeneration stage. Since the effect of RA is to proximalize positional information in blastema cells, these 11 proteins represent gene products that could be involved in a biochemical cascade leading to the establishment of positional information in the regenerating limb along this axis.


Visualization of a mammalian transcription initiation complex.

Various proteins required for the initiation of eukaryotic gene transcription by RNA polymerase II have been identified and characterized, but little is known about their organization into a functional unit. Here, we describe the appearance of the murine ribosomal protein (rp) L32 gene transcription initiation complex as determined by transmission electron microscopy. Using a fractionated nuclear extract enriched for transcription factors necessary for rpL32 gene transcription in vitro and a DNA fragment containing the rpL32 gene promoter, the transcription initiation complex was imaged by standard transmission electron microscopy. Quantitative image analysis demonstrated that the complex is a multilobed structure whose two-dimensional projections are approximately 24 x 34 nm in size. Looping of the DNA seen in these images suggests that the proteins residing at the promoter region associate with proteins several hundred base pairs distant to the RNA start site, with bending of the DNA allowing these interactions to occur.


Inhibitors of human immunodeficiency virus-1 protease.

We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) can be characterized by a high-affinity mode (Ki = 478 +/- 27 nM), resulting in pure competitive inhibition of the hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 microM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 +/- 1 microM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 microM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease.


Readthrough enhancement and promoter occlusion on the ribosomal genes of Xenopus laevis.

An RNA polymerase I termination site is found just upstream of the ribosomal gene promoter in mammals and amphibia. It has been suggested that this termination site may actively enhance ribosomal transcription in a process known as readthrough enhancement or that it may simply prevent the disruption of initiation complexes or promoter occlusion. There is, however, a consensus of opinion that the terminator is important for efficient ribosomal transcription. Here we have quantitatively investigated the relative importance of readthrough enhancement and promoter occlusion on the transcription of the microinjected Xenopus laevis ribosomal gene. The results show that, in this system, promoter occlusion is limited and terminator mutations predominantly affect readthrough enhancement. The terminator is shown to be unnecessary for the enhancer activity of the rest of the ribosomal spacer. Model calculations suggest that readthrough enhancement could be explained by polymerase recycling and that it may be unnecessary to postulate a specific mechanism of polymerase handover.


Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung.

Hydrophobic pulmonary surfactant protein enriched in SP-C has been mixed in amounts up to 10% by weight with various phospholipids. The lipids used were dipalmitoyl phosphatidylcholine (DPPC), or DPPC plus unsaturated phosphatidylglycerol (PG), or phosphatidylinositol (PI) in molar ratios of 9:1 and 7:3. The protein enhanced the rate and extent of adsorption of each lipid preparation into the air-water interface, and its respreading after compression on a surface balance. Maximum surface pressures attained on compression of monolayers of mixtures of lipids were slightly higher in the presence of protein. The effects on rate and extent of adsorption were proportional to the amount of protein present. Mixtures containing 30 mol% PG or PI adsorbed more readily into the interface than those containing 10% acidic lipid or DPPC alone. Mixtures containing 30% PI were slightly more rapidly adsorbed than those containing 30% PG. The results suggest that mixtures of DPPC with either acidic lipid in the presence of surfactant protein could be effective in artificial surfactants.


Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis.

The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70--chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to--100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to--188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP70 gene promoter can function during Xenopus embryogenesis.


The ability of bovine mammary tissue to synthesize lipids for 96 h when cocultured with liver and adipose tissue.

Explants of bovine mammary, liver, and adipose tissues were cocultured in the same well for 24, 48, 72 or 96 h and their ability to synthesize lipids was measured by the incorporation of [14C]acetate into triglycerides, free fatty acids, and phospholipids. The model was developed to study the effect of bovine somatotropin on lipid synthesis in mammary tissue, because somatotropin will not affect mammary tissue unless liver tissue is present. Somatotropin reduced incorporation by mammary tissue relative to the control. However, the rate of incorporation remained constant through 96 h with somatotropin, but steadily decreased in control mammary tissue. Phospholipid release into the media increased dramatically from 2% of that synthesized during the first 24 h to 218% of that synthesized from 72 to 96 h. The high release of phospholipids after 24 h suggests that cellular breakdown was occurring. A 24-h incubation is recommended to study lipid synthesis with this system.


Factor analysis of the Chinese WAIS with persons who have mental retardation.

The Chinese revision of the WAIS (designated WAIS-RC) was factor analyzed for a sample of individuals with mental retardation. There were 55 Chinese subjects with a mean Full Scale IQ of 49.13 (SD = 16.46). Results indicated that a two-factor solution comprised of the familiar Verbal Comprehension and Perceptual Organization factors best described the data. Comparison of the Chinese structure with that of a low-IQ Canadian sample tested with the American WAIS-R revealed congruence coefficients of .97 and .98 for Verbal Comprehension and Perceptual Organization, respectively. These findings provide support for further cross-cultural research utilizing the latest editions of the American and Chinese Wechsler scales.


Clinical effects of naltrexone on autistic behavior.

Eight young adults who were diagnosed with autism were given the opiate antagonist naltrexone to control self-injurious behavior and maladaptive idiosyncratic mannerisms. The drug and placebo were administered in a double-blind crossover design over 17 weeks. Although one subject appeared to have partial decreases in maladaptive behaviors associated with naltrexone use, the drugs, as administered in this study, did not reduce the self-injurious and other maladaptive behaviors of the subjects.


Predictors of response to antidepressant medications.

In a 10-year interval, 175 residents of a state institution who had severe or profound mental retardation were treated with heterocyclic antidepressants when other medications and forms of treatment had proved ineffective. Responders to the medication were identified and compared with nonresponders to determine which presenting symptoms and other parameters might serve as predictors of medication efficacy.


Haptic asymmetries in persons with and without mental retardation.

Adults with mild mental retardation and equal-MA children and adults without mental retardation were required to (a) tactually examine single letters, two-letter words, bigrams, and Chinese characters with their right or left index finger and (b) indicate whether a visually presented stimulus was the tactually examined stimulus by saying "same" or "different." The left hand was significantly better for "same" responses and the right hand, for "different" responses, suggesting that hemispheric processing is dependent on information-processing requirements rather than type of stimulus. A left hand advantage for "same" Chinese characters by the children and adults without mental retardation was due to an increase in right hand latencies rather than a decrease in left hand latencies, again suggesting that different types of analyses are employed by the two cerebral hemispheres. Adults with mental retardation had the poorest accuracy and slowest latencies for correct "different" responses and significantly less differentiation of "same" and "different" responses (A') for all stimulus types. They identified "same" letters, words, and bigrams significantly faster than Chinese characters with their left hand and showed a trend toward the differential processing of bigrams ("same" latencies) as a function of hand.


Attentional resource demands of stimulus encoding for persons with and without mental retardation.

Speed of encoding differences between individuals with and without mental retardation were examined to determine whether they stem from an automatically executed cognitive process or from encoding processes that require attentional resources. In Experiment 1, encoding functions were generated for physical identity and name identity encoding while subjects retained a full memory load or half memory load. Size of memory load influenced encoding times for all subjects. However, the pattern of group differences suggested that subjects with mental retardation allocated fewer attentional resources to encoding, even though encoding may require more of their resources for efficient execution. These conclusions were supported in Experiment 2, in which resource allocation was assessed using response times to auditory probes placed at various locations in the semantic encoding and decision task.


Localization of ras proto-oncogene expression during development in Xenopus laevis.

The expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S-labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.


Transcription of the H19 gene in differentiating cytotrophoblasts from human placenta.

Placental differentiation is closely correlated with the appearance of specific proteins, yet factors regulating cytotrophoblast differentiation are unknown. One strategy employed to search for such factors makes use of differential screening of cDNA libraries. For this purpose, cytotrophoblasts were isolated from human term placentae and cultured for 24 and 120 hr. cDNA libraries were constructed from the cell's RNA, and differential screening resulted in the isolation of three identical clones highly expressed after 120 hr. A DNA sequencing of 139 bp at the 3' end of these clones and a search of the data bank revealed that the sequence was identical to the parallel domain in the human H19 gene. This highly conserved gene is unusual in that it may not encode a protein. In the mouse, its RNA was shown to accumulate to high levels in embryonic tissues of endodermal and mesodermal origin. Our present findings imply that, in humans, the H19 gene is efficiently expressed in placental tissue and differentiated cytotrophoblasts, which are of ectodermal origin. RNA blot hybridization revealed a unique bimodal pattern of expression for the H19 gene in cultured cytotrophoblasts. The modulation in expression of H19 during cytotrophoblast growth was not due to the increase in the number of multinuclear cells. Size fractionation of cytotrophoblasts by centrifugal elutriation revealed that H19 expression is correlated with the stage of cell differentiation. We therefore propose that H19 transcripts might play a regulatory role in the process of cytotrophoblast differentiation.


Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: an immunocytochemical study.

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, Pl1, Pl2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of alpha-amanitin, the matrix morphology was altered for Pl1 and Pl2. alpha-Amanitin had no affect on the distribution of P1 or lamin B antigens. If alpha-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of Pl2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.


The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.


Golden hamster embryonic genome activation occurs at the two-cell stage: correlation with major developmental changes.

The earliest time of onset of embryonic genome activation in golden hamsters was investigated. The inhibition of transcription by alpha-amanitin (11 micrograms/ml) in cultured embryos resulted in a total arrest of development of early 2-cell embryos (26 hr post-egg activation); under similar conditions, immediate cleavage divisions of 1-, late 2-, 4-, and 8-cell embryos were not affected. Electrophoretic analysis of [35S]methionine-labeled embryonic proteins showed that alpha-amanitin treatment apparently inhibited transcription-dependent protein synthesis in early 2-cell and, to some extent, in late 2-cell when compared to 4-cell embryos. Analysis of total RNA synthesis, using [alpha 32P]-UTP or [32P]-orthophosphate, showed that there was a high proportion of radioactivity associated with the macromolecular fraction (RNA) at the early and late 2-cell stages and at the 4-cell stage compared to that at the 1-cell stage. These results indicate that the de novo synthesis of RNA, encoded by the embryonic genome, occurs at the 2-cell stage and that the second and subsequent cleavage divisions of hamster preimplantation embryos are dependent on new transcriptional activity. This initial activity of the embryonic genome in hamsters is coincident with several characteristic features of in vitro development such as a block to development, synthesis of major proteins, change in energy substrate preference, phosphate-inhibition of development and a requirement for amino acids.


Primary culture of human fallopian tube epithelial cells and co-culture of early mouse pre-embryos.

We have established a monolayer culture system for human fallopian tube epithelial cells. The cells were isolated from tubes using collagenase digestion, and were cultured in Ham's F-10 supplemented with 15% fetal calf serum. The epithelial cells derived from culture were characterized using immunocytochemical staining and electron microscopy. These cells were stained with antikeratin and anti-epithelial membrane antigen, but showed no staining after treatment with antivimentin. Electron microscopy showed many microvilli on the cell surface and tight junctions or desmosomes at areas of cell-cell contact. Cell proliferation was enhanced by epidermal growth factor, but not by fibroblast growth factor, insulin, transferrin, estradiol-17 beta, or progesterone. The 2-cell ICR mouse pre-embryos were co-cultured for 4 days with tubal epithelial cells (A) (n = 98), in cell-conditioned medium (B) (n = 83), or in medium alone (C) (n = 72). During the first 24 h in culture, for groups A and B, the rates of cleavage to the 4-cell stage were 90.9% and 81.9%, respectively. Cleavage rates in these two groups were significantly higher (P = 0.0012, P less than 0.00001) than in group C (56.9%). After 72 h in culture, the rates of development to the blastocyst stage were significantly higher for groups A and B compared to group C (89.6% and 73.5% vs. 54.5%, P less than 0.00001, P = 0.0002). These results suggest that factor(s) from tubal epithelial cells may facilitate the development of mouse pre-embryos throughout the pre-implantation stages.


Cleavage rate of diandric triploid mouse embryos during the preimplantation period.

The postimplantation development of human and animal triploid embryos is well documented, but there is little informative data on their preimplantation development. An analysis of cell number at appropriate times during this period and thus their cleavage rate would give an indication of the potential triploids have for further development and may explain some problems associated with their postimplantation development. To rule out any effects of technical procedures on cleavage rate, appropriate controls were used. Diandric triploid embryos were produced using standard micromanipulatory techniques, which involved the injection of a male pronucleus into a recipient one-cell-stage embryo. The karyoplast was fused to the cytoplasm by electrofusion, and the resulting tripronucleate diandric triploid embryos were transferred to appropriate pseudopregnant recipients. At specific times after the transfer, the embryos were recovered and cell numbers established. The results were plotted and regression lines drawn. Three controls were used 1) micromanipulated diploid embryos from which the male pronucleus had been removed and immediately reinserted and fused to restore diploidy, 2) diploid embryos that had been briefly incubated in cytochalasin D and colcemid to find out the effects these agents had on development, and 3) diploid embryos that had been isolated and briefly incubated in tissue culture medium. All embryos were subsequently transferred to recipients. After isolation at specific times during the preimplantation period, cell numbers were also established and the results plotted.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of electrofusion pulse in either electrolyte or nonelectrolyte fusion medium on subsequent murine embryonic development.

This study was designed to determine what effect electropulse parameters would have on rate of fusion, lysis, and embryo viability when embryos were subjected to electrofusion treatment in nonelectrolyte or electrolyte pulse media. Previous experiments have shown electrolyte medium (i.e., phosphate-buffered saline; PBS) to have a positive effect on electric pulse-induced murine oocyte activation. In addition, these results also indicated that pulse media containing 0.9 mM Ca2+ induced a dramatic increase in the rate of murine oocyte activation compared with oocytes pulsed in media containing 0.0 or 0.05 mM Ca2+. Pronuclear or two-cell-stage embryos were obtained from superovulated prepubertal randomly bred Swiss (albino) female mice. Embryos were randomly assigned to three nonelectrolyte and three electrolyte treatment media. Nonelectrolyte media consisted of 0.3 M mannitol (T1), 0.3 M mannitol + 0.05 mM CaCl2 (T2), and 0.3 M mannitol + 0.9 mM CaCl2 (T3). Electrolyte media consisted of Ca(2+)-free PBS (T4), PBS containing 0.05 mM CaCl2 (T5), and PBS containing 0.9 mM CaCl2 (T6). Three experiments were carried out; the objective of the first was to determine the rate of fusion and rate of lysis in murine two-cell embryos placed in the two types of (0.3 M mannitol, T1-T3; and PBS, T4-T6) fusion media and subjected to a fusion procedure (3 V, 5 sec AC alignment pulse, followed by a 1.56 kV.cm-1, 99 microsec DC fusion pulse). Control two-cell embryos were placed in T1 for 2 min and did not receive a fusion pulse.(ABSTRACT TRUNCATED AT 250 WORDS)


Morphological development and sex of bovine in vitro-fertilized embryos.

Bovine in vitro-fertilized embryos at the blastocyst stage were collected at days 7, 8, and 10 postinsemination and sex was determined via the polymerase chain reaction (PCR) to compare the embryonic development with the sex of the embryos. The percentages of males (sex ratio) after division of the embryos into three developmental groups were 68%, 48%, and 35% in the fast, intermediate, and slow groups, respectively (P = 0.014). The percentages of males on days 7, 8, and 10 were 60%, 40% and 33%, respectively (P = 0.043). The average sex ratio for the whole material was 50%. It is thus concluded that male bovine preimplantation embryos develop faster than female embryos.


Marsupial fertilization: some further ultrastructural observations on the dasyurid Sminthopsis crassicaudata.

Various morphological aspects of in vivo egg maturation and sperm-egg interaction were investigated in the Australian marsupial Sminthopsis crassicaudata with the transmission and scanning electron microscopes. Cortical granules invariably occurred in primary oocytes, with the number increasing after resumption of the first meiotic division. They generally occurred close to the oolemma, including the region near the oocyte nucleus. After mating, spermatozoa with intact acrosomes, which had a homogeneous electron-dense matrix, were found on the outer zona surface, but loss of acrosomal contents had occurred by the time of zona penetration. Sperm incorporation into the egg took place at the metaphase II stage of meiosis, and, at this time, cortical granules disappeared from the egg cortex. Sperm heads with condensed chromatin in the egg cytoplasm had an electron-dense layer of subacrosomal material over part of the dorsal nuclear surface, but no membranes were present around these incorporated spermatozoa. Sperm chromatin decondensation resulted in an elevation of egg cytoplasm, and the cell membrane over this area lacked microvilli. The pronuclear envelope was not laid down until after chromatin decondensation had occurred. By this time the fertilized egg had reached the uterus, and a smooth, electron-dense, shell membrane had been deposited. These observations, together with our previous findings, indicate that some of the processes of sperm-egg interaction are similar to those in eutherian mammals, whereas others appear highly divergent.


Synthesis and modification of D7 protein during Xenopus oocyte maturation.

The Xenopus maternal mRNA D7 is translationally repressed during oogenesis, only becoming recruited into polysomes during oocyte maturation, with D7 protein being detectable for the first time prior to germinal vesicle breakdown (GVBD). The synthesis of D7 protein was found to be induced by a variety of maturation-promoting agents including cyclin, c-mos and crude preparations of MPF. D7 protein induced by all these agents is post-translationally modified and exists as a number of variants of differing molecular weight. In contrast to endogenous D7 mRNA, D7 RNA injected into the stage VI oocyte is efficiently translated, resulting in the accumulation of predominantly unmodified D7 polypeptides, which become increasingly modified during oocyte maturation to produce a pattern of polypeptides similar to those derived from endogenous D7 mRNA. Thus, the system that results in the post-translational modification of the D7 protein is itself activated during oocyte maturation. The nature of the protein modification is not known but does not appear to be phosphorylation. The translation of exogenous D7 RNA in the stage VI oocyte does not lead to translational derepression of endogenous D7 mRNA.


Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.


Expression of interleukin-6 in porcine, ovine, and bovine preimplantation conceptuses.

A porcine interleukin-6 (pIL-6) cDNA has been cloned from pig spleen cDNA library to provide information that would allow us to study IL-6 mRNA expression during pregnancy of several domestic Artiodactyla. The cDNA is 1058 bp long and with a single open reading frame that encodes a 212 amino acid polypeptide with 28-residue signal sequence. It shares 61% and 43% amino acid sequence identity with human and mouse IL-6, respectively. PCR procedures with primers designed from regions of sequence conserved between human and pig have been used to identify IL-6 cDNA in lambda gt11 libraries constructed from day 15-16 (sheep), day 17 (cattle), and day 13-17 (pig) conceptus mRNA. The presence of IL-6 mRNA in elongating preimplantation ovine (days 13-25), porcine (days 13-21), and bovine (days 16-20) conceptuses was also demonstrated by PCR after reverse transcription of total ribonucleic acid with reverse transcriptase and by solution hybridization with a pIL-6 cRNA probe. These observations suggest that IL-6 is a product of these early conceptuses and may be involved in early maternal responses to the presence of an embryo within the uterus.


Drop out: a third chromosome maternal-effect locus required for formation of the Drosophila cellular blastoderm.

drop out (dop) is a recessive maternal-effect locus identified in a screen for female-sterile mutations in Drosophila polytene region 71C-F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularization in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.


Isolation of epiblast-specific cDNA clones by differential hybridization with polymerase chain reaction-amplified probes derived from single embryos.

A mouse day 7.5 embryonic ectoderm cDNA library containing 2 x 10(6) clones was screened by differential hybridization with polymerase chain reaction (PCR)-amplified probes derived from a single embryo. Day 7.5 ectoplacental cone and embryonic ectoderm served as the source of mRNA to make minus and plus probes, respectively. In a limited screen of fewer than 2,000 clones, 23 up-regulated clones were identified by the difference in hybridization signal with the two probes. DNA sequence analysis revealed the nature of some, but not all, of the clones. Northern blot and in situ hybridization with a subset of the clones confirmed the utility of the approach, since the differential signal was also observed in these experiments. This approach may prove useful for identifying genes that play a role during development.


Glycogen breakdown in cleaving Xenopus embryos is limited by ADP.

Xenopus eggs contain large stores of glycogen, but this glycogen is not glycolytically processed during cleavage. The Embden-Meyerhof pathway is inhibited by the absence of pyruvate kinase activity in vivo, and lactate and pyruvate are present at relatively low levels. In the late blastula, just preceding gastrulation, lactate levels increase, indicating the onset of glycogen breakdown and glycolytic flux. Glycolysis from microinjected [14C]glucose-6-phosphate could be transiently activated, however, by the coinjection of ADP into fertilized eggs, and constitutively activated by the injection of the ATPase potato apyrase, indicating the presence of all enzymes necessary for glycolytic activity. The isozyme profiles of pyruvate kinase and malic enzyme, two enzymes involved in carbon metabolism during cleavage or in the subsequent activation of glycogen breakdown, do not change between the egg and gastrula stages. These data suggest that the activation of glycogen breakdown and glycolysis in the late blastula is probably not a result of new gene activity but may be the metabolic consequence of increased free ADP that is then able to support the pyruvate kinase reaction.


Prefertilization treatment of mice with platelet activating factor affects pregnancy.

Embryo-derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development invivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.


Migration of centromere proteins in rabbit spermatids.

Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer-assisted three-dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelongating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centrometric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.


Purification and characterization of a vitelline coat lysin from Ciona intestinalis spermatozoa.

In Ciona intestinalis a chymotrypsin-like activity is involved in sperm penetration of the egg vitelline coat. A chymotrypsin-like enzyme has been purified from spermatozoa by a protocol including ion exchange chromatography, gel filtration, and native polyacrylamide gel electrophoresis. The purified enzyme resulted homogeneous when analyzed by SDS-PAGE. The molecular weight of the chymotrypsin-like enzyme was estimated to be 35 kDa by gel filtration and 24 KDa by SDS-PAGE in nonreducing conditions. The pH optimum of the enzyme is 8.4 and its activity is enhanced by Ca2+. It shows the highest activity towards the synthetic substrate Suc-Ala-Ala-Pro-Phe-AMC. Furthermore, by electron microscopy, the purified enzyme affects the structure of egg vitelline coat, and thus it fulfills one of the criteria of a lysin.


Collection of acrosome-reacted human sperm using monoclonal antibody-coated paramagnetic beads.

A monoclonal antibody (MAb) against human acrosome-reacted sperm was attached to paramagnetic polystyrene beads. Human sperm prepared by the swim-up method were 1) incubated in m-BWW, 2) incubated and ionophore treated, or 3) incubated with 5% seminal fluid. After treatment, sperm were mixed with the beads and incubated for 1 hr. Variously treated sperm showed different binding abilities to the beads. Sperm bound to the beads were collected by a magnet and subjected to triple staining. Most of the collected sperm were acrosome reacted. The results suggested that the beads can be used to estimate the acrosomal status of sperm, and that the use of antibody-coated paramagnetic beads provides a convenient way of collecting acrosome-reacted sperm. The acrosomal status detected by the beads was also compared with the ability of sperm to fuse with zona-free hamster eggs. It was found that greater bead-binding ability correlated with more sperm fusing with zona-free hamster eggs.


Evidence for differential maturation of reciprocal sperm segregants in the murine Rb(6.16) translocation heterozygote.

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3-day intervals, and aged sperm were studied after matings on 6-14 postoperative days. The sperm chromosome complement was analyzed in first-cleavage metaphase zygotes after sequential G- and C-banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P less than 0.001) from the expected 1:1. The ratio of X- to Y-bearing sperm also deviated from expected (P less than 0.01) mostly due to a significant deficiency (P less than 0.05) of balanced sperm that were X-bearing. Fertilized oocytes were recovered from matings of 10 males on days 6-8 postoperatively, and, of 139 metaphasic one-cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ratio in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation-bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function.


A rat sperm flagellar surface antigen that originates in the testis and is expressed on the flagellar surface during epididymal transit.

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin-biotin-peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X-100 extracts of epididymal sperm after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000-28,000 daltons (26-28K). Two-dimensional isoelectric focusing and SDS-PAGE indicated that the 26-28K antigen had an isoelectric focusing point (pl) of 5.8-5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26-35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit.


Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.


Laminar specificity of extrinsic cortical connections studied in coculture preparations.

The formation of specific neural connections in the cerebral cortex was studied using organotypic coculture preparations composed of subcortical and cortical regions. Morphological and electrophysiological analysis indicated that several cortical efferent and afferent connections, such as the corticothalamic, thalamocortical, corticocortical, and corticotectal connections, were established in the cocultures with essentially the same laminar specificity as that found in the adult cerebral cortex, but without specificity of sensory modality. This suggests the existence of a cell-cell recognition system between cortical or subcortical neurons and their final targets. This interaction produces lamina-specific connections, but is probably insufficient for the formation of the modality-specific connections.


A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.


Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.


Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.


Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo.

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.


Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons.

Opiates and the opioid peptide enkephalin can cause indirect excitation of principal cortical neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons. The mechanism by which opioids mediate these effects on interneurons is unknown, but enkephalin hyperpolarizes the somatic membrane potential of a variety of neurons in the brain, including hippocampal interneurons. We now report a new, more direct mechanism for the opioid-mediated reduction in synaptic inhibition. The enkephalin analog D-Ala2-Met5-enkephalinamide (DALA) decreases the frequency of miniature, action potential-independent, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) without causing a change in their amplitude. Thus, we conclude that DALA inhibits the action potential-independent release of GABA through a direct action on interneuronal synaptic terminals. In contrast, DALA reduces the amplitude of action potential-evoked, GABA-mediated IPSCs, as well as decreases their frequency. This suggests that the opioid-mediated inhibition of non-action potential-dependent GABA release reveals a mechanism that contributes to reducing action potential-evoked GABA release, thereby decreasing synaptic inhibition.


Functional expression and CNS distribution of a beta-alanine-sensitive neuronal GABA transporter.

The synaptic action of gamma-aminobutyric acid (GABA) is terminated by high affinity, Na(+)-dependent transport processes in both neurons and glia. We have isolated a novel GABA transporter cDNA, GAT-B, which encodes a high affinity (Km = 2.3 microM), Na(+)- and Cl(-)-dependent GABA transport protein that is potently blocked by beta-alanine, a compound generally considered a selective inhibitor of glial transport. However, in situ hybridization studies indicate that GAT-B mRNA is expressed predominantly within neurons. These data indicate that the neuronal-glial distinction of GABA transporters based on inhibitor sensitivities must be reconsidered and suggest a greater diversity of GABA transporters than has been predicted by previous pharmacologic studies.


Two cellular inductions involved in photoreceptor determination in the Xenopus retina.

Cellular determination in the Xenopus retina is not a strict consequence of cell lineage or cell birthdate. This suggests that a retinal cell gets its fate by either local cellular interactions, diffusible factors, or an indeterminate stochastic mechanism. We have performed an in vitro experiment in which cellular contact is controlled to test the first possibility directly. We use these experiments to demonstrate that two cellular inductions are involved in photoreceptor determination in vitro and that these inductions also occur during development in the retina in vivo. The first interaction is responsible for biasing cells toward either a generic photoreceptor or a cone fate, while the second directs cells toward a rod cell fate.


The matrix metalloproteinases and their inhibitors.

A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.


Effect of acute inflammatory lung injury on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat pulmonary alveolar macrophages.

Using a well-characterized rat model of immune complex-mediated acute inflammatory lung injury, we determined that there is a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Monocyte chemotactic activity is also significantly enhanced in culture supernatants from pulmonary alveolar macrophages (PAMs) from injured rat lungs. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein 1 (MCP-1) mRNA in PAMs obtained from rats with immune complex-induced lung injury. The increased expression of MCP-1 mRNA and associated increase in monocyte chemotactic activity present in culture supernatants of PAMs from injured rat lungs suggest that PAMs may participate in the pathogenesis of acute inflammatory lung injury by the secretion of monocyte chemoattractants including MCP-1.


Modulation of fibronectin production of bovine bronchial epithelial cells by transforming growth factor-beta.

Regulation of airway repair after injury is poorly understood but is thought to be important in the development of airway diseases such as chronic bronchitis and asthma. There is evidence that fibronectin (Fn), an extracellular matrix glycoprotein, has a role in repair processes. In addition, transforming growth factor-beta (TGF-beta) is also likely involved in would healing and is known to influence extracellular matrix constituents in other cell systems. We postulated that TGF-beta may effect airway repair by modulating Fn production from airway epithelial cells. To examine this hypothesis, we studied the effect of TGF-beta 1 on Fn production by bovine bronchial epithelial cells in culture. Fn, released into the media of cultures exposed to TGF-beta 1, increased in a dose- and time-responsive fashion. Fn in the cell layer also increased in response to TGF-beta 1. De novo protein synthesis was demonstrated by an increase in [35S]methionine incorporation into Fn immunoprecipitated from media of TGF-beta-treated cultures. TGF-beta 1 also induced an increase in expression of Fn mRNA from cultured bronchial epithelial cells, suggesting that TGF-beta modulates Fn production of these cells, at least in part, through modulation of Fn gene expression. These data support a role for TGF-beta in airway repair through modulation of Fn production by airway epithelium.


Transforming growth factor-beta is generated in the course of hypersensitivity pneumonitis: contribution to collagen synthesis.

Mice of the C57BL/6 strain were instilled with optimal doses (150 micrograms/day for 3 days/wk) of the thermophilic actinomycete Faeni rectivirgula (also known as Saccharopolyspora rectivirgula or Micropolyspora faeni) to induce a hypersensitivity pneumonitis inflammation that mimics the human disease affecting certain occupational groups. This mouse model was characterized by a very significant alveolitis (3-fold increase in bronchoalveolar lavage [BAL] cell number at 48 h and a 10-fold increase at 3 wk). Also, total lung transforming growth factor (TGF-beta) was shown to be elevated in treated mice as early as 1 wk after the first instillation and increased gradually to 2.5 micrograms/lung at 3 wk (approximately 0.3 microgram/lung in saline-instilled controls). Intranasal instillation with F. rectivirgula was also associated with very significant increases in lung fibroblast collagen synthesis, starting at 2 wk. BAL macrophages from mice instilled with F. rectivirgula were found to release significantly more TGF-beta upon in vitro stimulation with zymosan beads than did BAL macrophages from saline-instilled mice. Finally, we show that supernatants from activated BAL macrophages of mice given F. rectivirgula increased quite significantly collagen synthesis in normal mouse lung fibroblasts. This increase could be abrogated by treating conditioned medium with a rabbit antibody against TGF-beta. Collectively, these data suggest that TGF-beta is generated in the course of experimental mouse hypersensitivity pneumonitis and contributes significantly to collagen synthesis.


Characterization of the mucin differentiation in human lung adenocarcinoma cell lines.

Four human lung adenocarcinoma cell lines were established in serum-free F12 medium supplemented with insulin, transferrin, hydrocortisone, cholera toxin, selenium, epidermal growth factor, bovine hypothalamic extract, and retinoic acid. Histochemical analyses with periodic acid-Schiff with and without diastase treatment (PAS-D technique) and immunocytochemistry with a mucin-specific monoclonal antibody demonstrated that three of the cell lines (CL2, CL3, and NCL2) were capable of mucin production. Biochemical characterizations of mucin produced by adenocarcinoma cells were focused on one of the cell lines, CL2 cells, which showed the most prominent reactivity with mucin-specific monoclonal antibody. Biochemical analysis using the mucin precursors [3H]glucosamine and [14C]serine indicated that CL2 cells can synthesize high-molecular-weight (M(r) greater than 200 kD) glycoprotein molecules that can be immunoprecipitated by this mucin-specific monoclonal antibody. The high-molecular-weight glycoproteins isolated from CL2 cells specifically reacted with mucin-specific monoclonal antibody by Western blot analysis, and composition analyses showed high levels of serine and threonine and a low level of aromatic amino acids, which are similar to human airway mucin. These observations suggest that lung adenocarcinoma CL2 cells cultured in this serum-free medium can retain function of airway mucin synthesis. Cell kinetic studies of these four cell lines showed that the cell line (CL1) without the mucin differentiation had a higher proliferative index and a shorter population doubling time as compared with the other three cell lines (CL2, CL3, and NCL2) with mucin differentiation. Examination of the retinoblastoma protein expressions in these adenocarcinoma cell lines revealed a phosphorylated pattern that correlated inversely with the mucin synthesis status of these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)


Condensed tannin promotes the release of arachidonic acid from rabbit resident alveolar macrophages.

The condensed tannin present in cotton mill dust profoundly alters the functional capabilities of resident alveolar macrophages. Previous studies from this laboratory have shown that in vitro exposure of rabbit resident alveolar macrophages to condensed tannin significantly inhibits the ability of these cells to produce reactive oxygen intermediates or to ingest particles. In the present study, we demonstrate that condensed tannin also alters arachidonic acid (C20:4) metabolism in these cells. Exposure of rabbit resident alveolar macrophages to condensed tannin results in the time- and dose-dependent release of C20:4 from the membrane phospholipids. The release of C20:4 occurred only at tannin concentrations greater than 25 micrograms/ml and was maximal 90 min after the onset of exposure. The EC50 for release was 75 micrograms/ml. Exposure to 100 micrograms/ml tannin resulted in the release of 20 +/- 3% of the [14C]C20:4 incorporated in the cell membrane. In comparison, exposure to 160 micrograms/ml zymosan resulted in the release of 14 +/- 4% of the [14C]C20:4. For both tannin and zymosan, phosphatidylcholine and phosphatidylinositol were the principal sources of the released C20:4. Approximately 63% of the C20:4 released after zymosan stimulation was further metabolized, mainly via the cyclooxygenase pathway. The major metabolites were 6-keto-prostaglandin F1 alpha, prostaglandin F2 alpha, and prostaglandin E2. In contrast, only 24% of the C20:4 released by tannin was subsequently further metabolized. The metabolites formed were essentially evenly distributed between products of the cyclooxygenase pathway and the lipoxygenase pathway. Exposure of alveolar macrophages to 50 micrograms/ml tannin for 30 min reduced the ability of the cells to subsequently incorporate C20:4 by 50 to 70%. In contrast, exposure of the cells to 160 mg/ml zymosan for 30 min had only a minimal effect on the subsequent ability of these cells to incorporate C20:4. These results indicate that tannin promotes C20:4 release, at least in part, by inhibiting its reacylation back into phospholipids, a mechanism that differs from that of zymosan.


Dexamethasone-induced increase in platelet-derived growth factor (B) mRNA in human alveolar macrophages and myelomonocytic HL60 macrophage-like cells.

Platelet-derived growth factor (B) (PDGF(B)) from alveolar macrophages is thought to play a central role in orchestrating the fibrotic response. Because corticosteroids are widely used in the treatment of patients with lung fibrosis, we asked whether corticosteroids modulated PDGF(B) gene activation in macrophages. PDGF(B) mRNA in alveolar macrophages obtained from smokers was increased after culture in the presence of dexamethasone (P less than 0.05), interferon-gamma (IFN-gamma) (P less than 0.05), or both in combination (P less than 0.05). Dexamethasone did not alter the abundance of mRNA encoding transforming growth factor-beta (TGF-beta), but did decrease the mRNA of early growth response gene 2 (EGR2). These initial experiments required large numbers of cells and thus were performed on macrophages from smokers. The results were reproduced when PDGF(B) mRNA abundance in macrophages from healthy nonsmoking volunteers was measured by the reverse-transcriptase polymerase chain reaction (RT-PCR). There was an increase in PDGF(B) mRNA in macrophages from nonsmokers after stimulation with dexamethasone alone (P less than 0.05) or in combination with IFN-gamma (P less than 0.05). To provide adequate cell numbers for kinetic and dose-response studies, the in vitro model of phorbol ester (TPA)-induced differentiation of HL60 cells to macrophage-like cells was used. In these cells, dexamethasone caused a 20-fold increase in the abundance of PDGF(B) mRNA, which was concentration and time dependent but not associated with changes in TGF-beta or EGR2 mRNA. This study suggests that in addition to their anti-inflammatory effects, corticosteroids may also increase the abundance of PDGF(B) mRNA.


Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.


Centromeres and telomeres.

Centromeres and telomeres are both composed of specific DNA sequences and unique chromosomal proteins. Isolation and characterization of some of these sequences and proteins has greatly increased our knowledge of centromere and telomere structure. This information is allowing us to determine how centromeres and telomeres perform their various roles in a cell.


Cis and trans mechanisms of DNA repair.

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.


Chromatin changes during the cell cycle.

Considerable progress has recently been made in elucidating the biochemical mechanisms regulating changes in chromatin structure during all stages of the cell cycle. Although anticipated, the apparently ubiquitous role played by phosphorylation/dephosphorylation reactions in modulating these changes is, nonetheless, remarkable.


Nuclear envelope structure.

The past 18 months have seen significant advances in our knowledge of the constituents of the nuclear envelope, their interactions during interphase and the mechanisms involved in their mitotic dynamics. Although most of the new data are in general agreement with, and contribute detail to, our traditional image of the nuclear envelope, a few observations appear to mark the beginning of new and important directions in research.


RNA processing.

Significant progress has been made over the last year in our understanding of the roles that RNA-binding proteins play in pre-mRNA splicing, the components of the spliceosome and how these components relate to the mechanism of splicing. Of particular importance has been the sequence analysis of the first mammalian splicing factors and structural determination of an RNA-binding domain.


An acquired state: epigenetic mechanisms in transcription.

Even when we know the primary sequences and binding specificities of every transcriptional activator and repressor, our understanding of transcriptional regulation will be rudimentary. This is partly because major aspects of gene expression are governed by epigenetic mechanisms. These mechanisms may be responsible for apparently identical sequences being read by the transcriptional machinery in two different but heritable ways: active or repressed. Epigenetic transcriptional states have been encountered in a number of recent experiments.


Transcription factors and the control of DNA replication.

Initiation of DNA replication is mediated by the assembly of nucleoprotein complexes at cis-acting DNA sequences known as origins of replication. Recent studies in several systems show that accessory transcription factors accentuate origin utilization by multiple mechanisms. The remarkable similarities in the activities of accessory transcription factors at promoters and origins of replication suggest that transcription factors play a pivotal role in the regulation of chromosomal DNA synthesis in eukaryotic organisms.


Myc and Max: a putative transcriptional complex in search of a cellular target.

Biochemical and genetic observations have supported the hypothesis that Myc family proteins function to regulate genes important in cellular growth and differentiation. The recent findings that Myc proteins can associate with other cellular proteins, possess sequence-specific DNA-binding activity and may directly transactivate transcription of several candidate genes have provided an experimental framework in which to test the transcription factor model. Based on principles established for several well characterized viral oncoproteins, a model is presented in which the regulation of Myc function is controlled by specific cellular protein interactions that serve to activate or repress transactivation activity or deny access of the Myc complex to its target sequences.


New twists in class III transcription.

Recent work emphasizes the similarity between polymerase II and III in the mechanisms of transcription. Highlights of the past year include the alignment of individual polypeptides within class III transcription complexes and the demonstration that class III transcription machinery includes TFIID and an RNA component.


Transcription factors: positive and negative regulators of cell growth and disease.

Recent advances in our understanding of transcription factors that regulate RNA polymerase II transcribed genes are described. This review considers the role of alternate protein-protein interactions to yield positive or negative regulation, the consequences of deregulated factor activity on cell function and approaches to establish factor-binding specificities.


Advances in RNA polymerase II transcription.

Multiple protein factors are necessary to mediate transcription by RNA polymerase II. Recently, a number of advances have been made in our understanding of how general transcription factors collectively modulate basal transcription in the context of different promoter environments and how this process is activated and repressed by accessory components.


Signal transduction: the nuclear target.

Fos and jun heterodimers activate the transcription of genes containing an AP-1 site. The activity of Fos and Jun proteins is regulated by post-translational modification. The activity of the rel/NF-kappa family of transcriptional factors is regulated by their sequestration in the cytoplasm in association with the inhibitor protein, I kappa B. An ankyrin repeat motif in I kappa B proteins is required for their direct association with rel/NF-kappa B.


Strut fracture with Björk-Shiley 70 degrees convexo-concave valve. An international multi-institutional follow-up study.

Between 1980 and 1983, 831 Björk-Shiley 70 degrees convexo-concave prosthetic heart valves were implanted at five institutions in Sweden, Germany. The Netherlands, and Canada. As of January 1991, there were 34 outlet strut fractures occurring from 0.2 to 10.1 years (median = 4.6 years) after implantation. In addition, there were 28 sudden, unexplained deaths. The mortality after strut fracture was 84%. The mortality after emergency valve replacement for strut fracture was 50%. The 10-year actuarial fracture rate (standard error) was 10.5 (2.4)% for large (29-33 mm) valves vs. 3.3 (1.2)% for 21-27 mm valves (P less than 0.001). Within valve size groups, fracture rates for aortic and mitral valves were similar. Cox regression analysis found only valve size to be significantly associated with strut fracture. There is a further subgrouping of the valves according to the manufacturer: group I are the earlier large 29-33 mm) valves; group II are the later large valves; group III are the small size (21-27 mm) valves. The risk of strut fracture was highest in group I (12.3% at 10 years) with an approximatively constant hazard (1.4% per year). A comparison was made with a statistical model incorporating all cases reported to the manufacturer. This model estimates fracture rates approximately 63%-73% of those found in the present study. These findings lead us to recommend that group I patients should be considered for elective reoperation on an individual basis, giving careful attention to risk factors and contraindications.


An appraisal of patch enlargement of the small aortic annulus in 33 patients undergoing aortic valve replacement.

To avoid the problem of valve prosthesis/patient mismatch in the late postoperative period, we have adopted patch enlargement of the small aortic annulus in 33 patients having undergone aortic valve replacement since 1975, when the aortic annulus could not accept a large enough prosthesis. The mean age was 40 years, including 2 children aged 4. The technique of patch enlargement utilized was Nicks' method wherein the incision does not extend onto the mitral leaflet. In 31 adult patients, the aortic annulus before enlargement ranged in diameter from 18 to 24 mm with an average of 20.8 mm. After enlargement, the diameter of the annulus was 25.0 mm on average. In 2 children, the mean diameter of the annulus was enlarged from 16.5 mm to 20.5 mm. There were 2 early deaths in the initial series and 2 late deaths. Patch enlargement of the small aortic annulus is a simple, safe, and effective adjunct permitting the insertion of a valve one or two sizes larger than that which could be accommodated by the native annulus.


The hydrodynamic function and leaflet dynamics of aortic and pulmonary roots and valves: an in vitro study.

Fresh homograft aortic and pulmonary roots were tested in a pulsatile flow simulator to assess their hydrodynamic function and leaflet opening characteristics. Simultaneous flow and pressure measurements were obtained for a range of cardiac outputs. The effective orifice area and regurgitant volumes were calculated. The mean pressure difference across the pulmonary roots was obtained under both left and right side pressures. A video recording of valve leaflet function was also obtained for each valve. A comparison was made with four porcine bioprosthetic heart valves (21 and 23 mm). The mean pressure difference with respect to flow for the 21 mm Hancock II and Intact porcine bioprostheses was significantly higher than that for all aortic roots tested. The 24 mm aortic root showed significantly lower pressure drop compared to all porcine valves tested. The mean pressure difference across each pulmonary root at pulmonary pressures was significantly greater than at systemic pressures. At systemic pressures the fully open leaflets had a triangular orifice with low leaflet open bending strains at the commissures. At the lower internal pressures, with reduced dilation of the root, higher bending strains were noted. These were not as severe as seen in porcine valves.


Plication of the diaphragm for unilateral eventration or paralysis.

Unilateral diaphragmatic paralysis and eventration have the same appearance and provoke the same disturbances. Diaphragmatic plication is intended to decrease lung compression, to make the thoracic base and mediastinum more stable, and to strengthen the respiratory action of intercostal, perithoracic, and abdominal muscles: 13 infants and children were operated upon, 7 in acute respiratory failure and ventilator-dependent, 4 in chronic respiratory failure; 11 adults were operated upon, 8 with respiratory and 3 with digestive symptoms. Four infants who had been operated upon before the 10th day of life died: 3 from associated diseases and 1 from a lung infection. The 9 survivors have been followed up for a mean period of 6.6 years. All were asymptomatic and the position of the plicated diaphragm was maintained. The 11 adults have been followed up for a mean period of 8.5 years. Nine were asymptomatic; in 1, dyspnea had decreased; in 1, reflux persisted and was surgically cured. In 5 adults, the respiratory tests showed a mean amelioration of 20% of vital capacity and 15% of forced expiratory volume in 1 s. In infants, the prognosis depends on associated malformations and on the condition of the lung. Plication should be performed after 2 weeks on a ventilator. In older children and adults, plication is justified when the anomaly produces symptoms (malignancy excluded). Plication is simple, efficient, and durable, but there is no indication of subsequent diaphragmatic function: its effects on respiratory mechanics are probably indirect.


Mediastinoscopy vs. anterior mediastinotomy in the diagnosis of mediastinal lymphoma: a randomized trial.

We selected 95 patients with mediastinal adenopathy and no signs of goiter, myasthenia gravis or mediastinal involvement by other disease. All patients underwent, for screening purposes, transthoracic fine needle aspiration biopsy based on chest x-ray and CT findings. Patients were then subdivided into 4 groups. One group of 22 patients with prevalent anterior mass localization underwent anterior mediastinotomy. One group of 19 patients with prevalent middle mediastinal mass localization underwent cervical mediastinoscopy. Two other groups of 27 patients each with both anterior and middle mediastinum localization randomly underwent anterior mediastinotomy or mediastinoscopy. Fifty-one Hodgkin's and 44 non-Hodgkin's lymphomas were diagnosed in total. In 11 cases (11.57%), median sternotomy (2) or thoracotomy (9) were necessary for establishing the final diagnosis. The overall diagnostic accuracy was 80.43% for cervical mediastinoscopy and 95.91% for anterior mediastinotomy. The statistical analysis performed on all patients showed a significant difference (chi 2 = 5.56, P less than 0.025, df = 1) between the two procedures.


Intermediate to late results of surgical relief of vascular tracheobronchial compression.

Since 1979, a total of 69 infants and children aged 0.1 to 11.9 (median 0.7) years required surgical intervention for: double aortic arch (26), anomalous origin of innominate artery (26), right aortic arch with left ligamentum arteriosum (9), pulmonary artery sling (5), retroesophageal right subclavian artery (3). Before operation, the morbidity was high: 84% of children were symptomatic in the first trimester of life with 24 patients requiring mechanical ventilation. Seven children had an accompanying heart defect. Left thoracotomy was the preferred approach. There were 2 early (asphyxic brain damage, postoperative pneumonia) and 2 late (tracheomalacia, complex heart defect) deaths. Five patients (2 originally operated elsewhere) needed reoperation for persistent symptoms. All 65 survivors are well 1 month to 11.9 (mean 3.9 +/- 3.62) years after surgery. Tracheobronchoscopy, magnetic resonance imaging, and lung function testing were helpful for postoperative evaluation. Minor tracheal compression was revealed in 4 patients despite their good clinical condition.


Myocardial oxygen consumption and lactate production during antegrade warm blood cardioplegia.

Continuous warm blood cardioplegia has recently been recommended as an alternative to multidose cold blood cardioplegia for myocardial protection during coronary bypass operations. Cardioplegia may have to be interrupted in order to provide a bloodless operating field during coronary anastomosis. To determine the effects of ischemia at normothermia on myocardial oxygen consumption and lactate production we randomized 17 dogs to receive either warm blood cardioplegia (37 degrees C) or cold blood cardioplegia combined with systemic and topical cooling. After initiating arrest, cardioplegia was interrupted for periods of 1, 2, 3, 4, 5, 6, and 10 min. Myocardial oxygen debt occurred after 3.5 min of ischemia in the 9 animals receiving warm blood cardioplegia. In contrast, myocardial oxygen consumption never exceeded oxygen availability during cold blood cardioplegia (P less than 0.001). Lactate production increased linearly in both groups but was much greater in those animals receiving warm blood cardioplegia (P less than 0.001). Spontaneous electromechanical activity was much more common during warm blood cardioplegia which required frequent infusions of cardioplegia to maintain cardiac arrest (P less than 0.0003). (1) Oxygen debt occurred after 3.5 min of warm ischemia; (2) spontaneous electromechanical activity is more common during warm heart protection which necessitates the use of larger volumes of cardioplegia to maintain cardiac arrest.