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PubMed1992

Gallbladder stones--dissolve, blast, or extract? Laparoscopic cholecystectomy versus 'the rest'.

This article reviews selected aspects of the non-surgical/minimally invasive treatments of gallbladder stones (GBS) and discusses briefly the residual role of these treatments in the era of laparoscopic cholecystectomy. In patients with specific, gallstone-related symptoms who wish to retain their 'functioning' gallbladders, there are at least six different management options. They range from rapid but invasive to slow but safe: i) the rotary lithotrite; ii) percutaneous cholecystolithotomy; iii) percutaneous transhepatic or iv) endoscopic retrograde cannulation of the gallbladder followed by instillation (manual or pump-assisted) of contact solvents; v) extracorporeal shock-wave lithotripsy + adjuvant bile acids and; vi) oral bile acids alone. The recommended investigation sequence is i) ultrasound (to diagnose the presence of GBS), followed by ii) oral cholecystography (to assess cystic duct patency, gallbladder anatomy and GBS size, number, lucency, buoyancy, and contour), and iii) regional computed tomography scanning of the gallbladder (to predict stone composition and dissolvability and to plan routes of access to the gallbladder). The decision-making steps are i) choice of some form of active treatment versus no treatment (other than observation); ii) in those with specific symptoms and a patent cystic duct who opt for active treatment, to choose between removing versus retaining the gallbladder; and iii) in those who wish to retain their 'functioning' gallbladder, to offer and select the most appropriate of the alternative options. In conclusion, despite the excellence of laparoscopic cholecystectomy, there remains a place for the non-surgical/minimally invasive approaches in a carefully selected minority of symptomatic GBS patients. Although GBS may recur in approximately 50% of patients, the recurrent stones are often asymptomatic, can be detected 'early' by follow-up ultrasound, and are easily treated. Ultimately, the aim of gallstone research must be to prevent not only recurrent but also primary GBS formation, which would obviate the need for both medical and surgical treatment.

Administration, Oral↗

PubMed1989

A two-step timed sequential treatment for acute myelocytic leukemia.

Since 1980, adults with acute myelocytic leukemia (AML) have been treated on two clinical studies using intensive timed sequential therapy. All patients ages 16 to 80, including those with secondary AML (SAML) and those with AML preceded by a hematologic disorder (AHD), were treated, regardless of medical complications at the time of diagnosis. The first study combined high doses of cytarabine (ara-C, AC) and daunorubicin (DRN, D) in sequence (Ac2-D-Ac) and resulted in a complete remission rate of 55%. A group of these patients selected by functional status was able to receive a second course of therapy in remission, which resulted in a disease-free survival (DFS) of greater than 40% at 7 years. Because of toxicity in that study, 114 patients were entered on a second trial initiated 4 years ago, using a less aggressive first course, with amsacrine, to achieve a stable remission (Ac2-D-Amsa). This first treatment was followed by a more intensive second course (Ac6-D-Ac). With this two-step approach, a higher complete remission (CR) rate (76% for de novo AML and 54% for SAML-AHD) was achieved, and more patients were able to receive the second course of therapy. At the current median follow-up of 26 months, the median duration of DFS and overall survival are 11 and 14 months for patients with de novo AML. Age less than or equal to 55 is the most significant prognostic factor for both prolonged DFS and overall survival, with median durations of 17 and 18 months, respectively, for these younger patients. Patients with SAML-AHD remain relatively refractory to treatment despite aggressive chemotherapy, with median durations of DFS and overall survival of 9 months and 5 months, respectively.

Adolescent↗

PubMed1989

Synthesis and adsorption of a poly(N-acetylethyleneimine)-polyethyleneoxide-poly (N-acetylethyleneimine) triblock-copolymer at a silica/solution interface. Influence of its preadsorption on platelet adhesion and fibrinogen adsorption.

The synthesis of a triblock copolymer poly-(N-acetylethyleneimine)-polyethylenoxide-poly(N-acet ylethyleneimine) includes two successive steps: the first is the functionalization of a poly(ethyleneglycol) precursor by creating sulfonic esters at its chain ends, the second uses these esters to initiate the cationic polymerization of 2-methyl-2-oxazoline. Homopolymers appear in the raw product; hence successive selective extractions of the copolymer with benzene and dioxane are necessary. The final yield in pure copolymer was 11%. The copolymer was characterized by UV and 1H-NMR spectrometry and light scattering. Adsorption isotherms were determined on silica, for varying pH and salt concentration. Optimum conditions for coating silica with the polymer were determined. The efficiency of this precoating to reduce the adsorption of fibrinogen was very high (99.2% reduction with respect to bare silica). Steric exclusion chromatography of a variety of proteins gave a satisfactory calibration curve. Platelet accumulation on copolymer precoated glass was reduced to 10-20% of its value on bare glass, a result superior to that obtained by albumin passivation of the same glass surface.

Adsorption↗

PubMed1990

Targeted gene replacement at the endogenous APRT locus in CHO cells.

We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation. Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.

Adenine↗

PubMed1992

Regularized linear method for reconstruction of three-dimensional microscopic objects from optical sections.

The inverse problem involving the determination of a three-dimensional biological structure from images obtained by means of optical-sectioning microscopy is ill posed. Although the linear least-squares solution can be obtained rapidly by inverse filtering, we show here that it is unstable because of the inversion of small eigenvalues of the microscope's point-spread-function operator. We have regularized the problem by application of the linear-precision-gauge formalism of Joyce and Root [J. Opt. Soc. Am. A 1, 149 (1984)]. In our method the solution is regularized by being constrained to lie in a subspace spanned by the eigenvectors corresponding to a selected number of large eigenvalues. The trade-off between the variance and the regularization error determines the number of eigenvalues inverted in the estimation. The resulting linear method is a one-step algorithm that yields, in a few seconds, solutions that are optimal in the mean-square sense when the correct number of eigenvalues are inverted. Results from sensitivity studies show that the proposed method is robust to noise and to underestimation of the width of the point-spread function. The method proposed here is particularly useful for applications in which processing speed is critical, such as studies of living specimens and time-lapse analyses. For these applications existing iterative methods are impractical without expensive and/or specially designed hardware.

Computer Simulation↗

PubMed1986

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.

Animals↗

PubMed1990

Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription.

All reactions involving reverse transcription of RNA are segregated from the cytosol within a subviral particle or capsid composed of the major capsid protein, the polymerase and the RNA template. A key step in the formation of these particles is the selective encapsidation of the RNA template. Although an important general feature of the reverse transcription pathway, encapsidation has been carefully studied only for retroviruses. We have now examined the encapsidation reaction in a family of enveloped DNA viruses that replicate by reverse transcription--the hepatitis B viruses (hepadnaviruses). Our results indicate that the hepadnaviral polymerase (P) gene product is required for RNA packaging, and that the encapsidation function of the enzyme can be separated from its DNA polymerase activity. To our knowledge, this is the first description of a role for polymerase gene products in this step of the reverse transcription pathway.

Amino Acid Sequence↗

PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗

PubMed1989

Nutritional and hormonal regulation of the gene for avian malic enzyme.

This paper reviews work from our laboratory on the molecular mechanisms involved in the nutritional and hormonal regulation of avian malic enzyme. The activity of hepatic malic enzyme, one of the set of "lipogenic" enzymes, is high in well-fed chickens and low in starved chickens. In chick embryo hepatocytes in culture, insulin and triiodothyronine (T3) are positive effectors and glucagon, acting via cyclic AMP, is a negative effector. Hormone concentrations in blood are consistent with insulin and T3 playing the major positive roles, and glucagon a major negative role, in regulating hepatic malic enzyme activity during the transitions between the fed and the starved states. New results indicate that insulin-like growth factor 1 also stimulates accumulation of malic enzyme. Our strategy has been to trace the intracellular signalling pathway from its distal end, altered enzyme activity, towards its proximal end, interaction of humoral factors with their appropriate cellular receptors. Nutrition- and hormone-induced changes in malic enzyme activity are due to altered concentrations of malic enzyme protein which, in turn, are due to altered rates of synthesis of malic enzyme. Synthesis of malic enzyme is controlled by regulating the level of malic enzyme mRNA which, in turn is regulated at initiation of transcription. The next step in this analysis will be to identify cis-acting sequence elements in the malic enzyme gene which bestow upon it a selective response to nutritional state and hormones. We are using transient expression systems and avian retroviral vectors to test the function of cis-acting elements involved in the regulation of transcription.

Animal Nutritional Physiological Phenomena↗

PubMed1986

Selective phagocytosis of gram-positive bacteria and interleukin 1-like factor production by a subpopulation of large granular lymphocytes.

There has been a consensus that a large granular lymphocyte (LGL) population with natural killer (NK) function is nonadherent and nonphagocytic. However, a significant proportion of the nonadherent cells purified by the two-step depletion of adherent cells with a plastic surface and nylon wool columns engulfed Sta. aureus into their cytoplasm. These cells were morphologically identified as LGL in light and electron microscopies. Two-color immunofluorescence tests, furthermore, demonstrated that Leu-11+ LGL, Leu-11+7-, and Leu-11+7+, but not Leu-11-7+, phagocytosed Sta. aureus. Among the particles tested here, only Gram(+) bacteria were preferentially phagocytosed, whereas Gram(-) bacteria, other large-sized microbes (e.g., baker's yeast and Candida albicans), latex, silica, and carbonyl iron were not. LGL exhibited a substantial level of bactericidal activity against Sta. aureus, although the level was one third of that mediated by monocytes. When Gram(+) bacteria were incubated with nonadherent cells for 18 hr, significant amounts of interleukin 1 (IL 1)-like factors (or IL 1 itself) as well as interferon were detected in the supernatants. On the other hand, this incubation did not induce interleukin 2 (IL 2). The IL 1-like factor producer cells were demonstrated to be the low-density lymphocytes on Percoll separation and to have the Leu-11+ phenotype. The phagocytosis was suggested to be an important stimulus in producing IL 1-like factors from LGL. Thus, the treatment of cells with cytochalasin B, a microfilament disrupting agent, completely abrogated both phagocytosis and IL 1-like factor production. Some cell wall components of Gram(+) bacteria might be important to a recognition process of the phagocytosis, since the protoplasts of Sta. aureus, when prepared by the treatment of bacteria with lysostaphin, were no longer phagocytosed by LGL. The present results therefore identify an additional unique characteristic similar to, but not identical with, the myelomonocytic nature of Leu-11+ LGL.

Adult↗

PubMed1992

Implementing a regulation-complaint quality improvement program on a commercial laboratory information system.

Implementing a quality improvement (QI) program on an automated laboratory information system (LIS) in the current regulatory climate requires first that the QI program be defined and second that the selected LIS be able to capture important events and use flexible vendor-provided or user-defined routines to prepare reports. Reports key on specific monitors and thresholds defined in the QI program. The product of a pathology laboratory is communicated information. The QI program focuses on the accuracy, clarity and timeliness with which the whole information-generating process functions. To support peer review the LIS must be able to select reports for evaluation based on user-defined parameters, such as diagnosis keyed through Systematized Nomenclature of Medicine codes, or by random or pattern selection by accession number. Counting and review of revised reports will focus attention on accuracy and skill in communication since these indicators often reflect client satisfaction with the report. To link services--e.g., cytology with surgical pathology--the LIS must be able to gather cases from the accession lists of both services and to flag diagnostic inconsistencies. LIS transaction logging at every step in the information process allows tracking of work load, productivity and resource utilization by functional areas and by individual, thus meeting regulatory requirements. Transaction logging also provides management information, such as segmented turnaround time audits, pinpointing sources of delay by kind and location of work or individual involved. Critical data must be held on-line for at least five years.

Clinical Laboratory Information Systems↗

PubMed1991

High level expression of recombinant human tissue factor in Chinese hamster ovary cells as a human thromboplastin.

Tissue factor (TF) is the high affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades by factor VIIa. We describe the synthesis of recombinant huTF by stably transfected CHO cell lines carrying integrated huTF DNA, and the isolation of huTF glycoprotein with specific functional activity equivalent to natural huTF. The expression vector (pCDM8), carrying the cytomegalovirus promoter to drive transcription of a partial cDNA construct encoding the complete huTF protein chain, was cotransfected with a plasmid containing the neomycin resistance gene for selection. These clones were further selected for level of expression of huTF protein. Optimal expression compatible with stability and cell growth was approximately 13.5 x 10(6) molecules per cell. To our knowledge, this is one of the highest levels of expression described for a recombinant transmembrane receptor in mammalian cells. Recombinant huTF protein was obtained by single-step immuno-affinity purification, and exhibits heterogeneity due to N-linked glycosylation. The protein was indistinguishable from natural huTF based on functional properties of the glycoprotein reconstituted in lipid vesicles, and expression of conformational epitopes. Large scale production of recombinant huTF is feasible to permit basic studies of protein structure as well as for design of huTF thromboplastin reagents.

Animals↗

PubMed1975

Gene duplication as a mechanism of genetic adaptation in Saccharomyces cerevisiae.

It has been shown that specific mutations of the gene that codes for the general acid monophophatase (Aphtase) of S. cerevisiae can increase the affinity of this enzyme for beta-glycerophosphate (BGP) and thereby provide this organism with the capacity to exploit extremely low concentrations of this organic phosphate (Francis and Hansche 1973). In this report two additional avenues are demonstrated to be available to this organism for increasing its capacity to exploit low concentrations of organic phosphates. One avenue is through mutations that increase the amount of Aphtase that associates with the cell wall, where it catalizes the hydrolysis of exogenous organic phosphates. The other avenue is through duplication of the gene that codes for Aphtase, doubling the amount of Aphtase synthesized.--The spontaneous duplication of the structural gene of Aphtase and the incorporation of the duplicate into this experimental population as a means of exploiting low concentrations of exogenous organic phosphates provides direct support for the first step of the mechanism through which new metabolic functions are postulated to evolve.

Acid Phosphatase↗

PubMed1985

Differential modulation of the expression of axonal proteins by non-neuronal cells of the peripheral and central nervous system.

Axonal behavior during the formation of the neuronal network of the nervous system has been shown to be under environmental control. Hence, as a first step in a project aiming to elucidate the molecular basis of axonal functions, we have identified axonal proteins whose synthesis is subject to environmentally induced changes. Neurons from chicken embryonic dorsal root ganglia (DRG) were grown in a compartmental cell culture system that allows selective examination of axonal proteins. Non-neuronal cells of the peripheral or central nervous system were co-cultured with the DRG axons. The axonal proteins expressed under these different environmental conditions were examined by metabolic labeling and two-dimensional SDS-polyacrylamide gel electrophoresis. Computerized quantification revealed that 12 out of 400 axonal proteins responded to changes in the local axonal environment by a change in their relative abundance. Some proteins changed in response to both types of co-cultures whereas some changed specifically under the influence of either peripheral or central non-neuronal cells.

Animals↗

PubMed1986

A cloned T cell line that selectively augments antibody responses of phosphorylcholine-specific B cells bearing the T15 idiotype.

Helper T (Th) cells have shown to be heterogeneous both in their function and in their specificity. Several studies have demonstrated that there exist Th cells which do not appear to be specific for antigen:Ia complexes (not H-2 restricted) but interact with the target B cell via recognition of immunoglobulin idiotypic determinants. In analyzing anti-phosphorylcholine (PC) responses, such idiotype-specific Th cells are characterized by 1) their ability to increase responses to phosphorylcholine by augmenting selectively the activation of T15-idiotype-bearing PC-specific B cells, 2) their ability to bind specifically to T15-bearing immunoglobulin and 3) the dependence of their maturation on circulating T15 idiotype rather than Ia. Our aim is to explore further the relationship between Ia-specific and T15-specific Th cells. These studies describe the first step in comparing specificity and function of idiotype-specific Th cells to other known Th cells by the cloning of a T15-specific Lyl T cell (ThId) with properties consistent with those described for uncloned populations. The cloned ThId cells generated do not activate B cells to secrete antibody but augment anti-PC plaque forming cell (PFC) responses induced by Ia-restricted Th cells. This augmentation is seen only in the T15-bearing PFC responses even though non-T15-bearing B cells are equally accessible, and it is seen only in PC responses and not responses to TNP under identical culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Isolation of highly purified lymphocyte subsets for functional studies by means of an indirect rosette technique.

An indirect rosette assay, utilizing ox erythrocytes (RBC) coupled with rabbit anti-mouse IgG and lymphocytes sensitized with monoclonal mouse antibodies against membrane markers, was used for purification of lymphocyte subsets that were functionally intact. Either peripheral blood mononuclear cells (PBMC) or T lymphocytes isolated by sheep RBC rosetting could be used as starting material for obtaining pure T-cell subsets (T4 or T8). The following steps of the method were evaluated: the procedure of coupling rabbit anti-mouse IgG to ox RBC via the CrCl3 method, the experimental conditions for specific rosetting, and the use of Percoll for the separation of rosettes from the non-rosetting cells. Under optimal experimental conditions the recovery of positively selected cells was 45-55% of the cells originally present in the PBMC. The purity of these cells reached a value of more than 95%, whereas the contamination of the depleted fraction was less than 3%. The functional integrity, manifesting itself as proliferation after mitogen stimulation and as regulatory influences on in vitro Ig synthesis, appeared to be unimpaired. The described technique may be applied to the purification of various cell subpopulations for functional studies, provided monoclonal antibodies against membrane antigens are available.

Animals↗

PubMed1992

Involvement of the N-methyl D-aspartate (NMDA) receptor in synapse elimination during cerebellar development.

In many instances, the establishment of highly specific neuronal connections during development results from the rearrangement of axonal projections through the trimming of exuberant collaterals or the elimination of functional synapses or both. Although the involvement of the N-methyl D-aspartate (NMDA) subtype of the glutamate receptor has been demonstrated in the shaping of axonal arbors, its participation in the process of selective stabilization of synapses remains an open issue. In this study, the effects of chronic in vivo application of D,L-2-amino-5-phosphonovaleric acid (D,L-APV), a selective antagonist of the NMDA receptor, on the synapse elimination process that takes place in the developing cerebellum of the rat have been analyzed. D,L-APV treatment prevented the regression of supernumerary climbing fiber synapses in 49 percent of the recorded Purkinje cells, while the inactive isomer L-APV was ineffective. Thus, activation of the NMDA receptor is a critical step in the regression of functional synapses during development.

2-Amino-5-phosphonovalerate↗

PubMed1989

Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Expression of the human immunodeficiency virus tat-encoded protein (Tat) is required for virus replication. A genetic approach was used to facilitate the purification of biologically active Tat. A recombinant Tat protein containing a stretch of six histidine residues and a protease cleavage site was engineered and purified to greater than 95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity resin that has selectivity for proteins with neighboring histidine residues. A modified scrape loading method for introduction of protein into cell monolayers was used to demonstrate that the purified Tat retained biological activity. Tat function was completely blocked in the presence of transcription inhibitors, which demonstrates the requirement of ongoing mRNA synthesis for trans-activation. These studies indicate that the mechanism of trans-activation is unlikely to involve a direct action of Tat on mRNA stability, transport, or translation and provides the basis for a rapid assay that can be used to identify inhibitors of trans-activation. The methods described herein should be useful for the functional analysis of other proteins that do not confer activity through a receptor-mediated pathway.

Amino Acid Sequence↗