Biomedical subjects

K Adler

Publications and source records attributed to K Adler.

At least 19 recordsLinked to original sources

PubMed1999

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.

Journal Article↗

PubMed1998

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation.

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.

Animals↗

PubMed1998

Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain.

The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

Amino Acid Sequence↗

PubMed1998

Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution.

The tissue-specific expression pattern and the intracellular distribution of the Ca(2+)-binding protein calreticulin at the mRNA and protein levels have been studied during somatic and zygotic embryogenesis of Nicotiana plumbaginifolia Viv. A full-length cDNA sequence encoding calreticulin was isolated from a lembda Zap cDNA library from early developmental stages of somatic embryogenesis. The deduced amino acid sequence of the calreticulin from N. plumbaginifolia shows high homology to the corresponding proteins of tobacco (98.2% identity), maize (80%) and barley (76.5%), and more than 55% homology to animal calreticulins, and the sequence motifs with established functions found in calreticulins of other species were quite conserved. Northern experiments revealed a developmental regulation of the calreticulin transcript with a maximum during the early stages of somatic embryogenesis and an auxin dependence during in-vitro cell culture. alpha-Naphthaleneacetic acid stimulated calreticulin expression whereas 2,4-dichlorophenoxyacetic acid reduced it. Immunohistological analysis of calreticulin distribution in the ovaries during zygotic embryogenesis showed that calreticulin biosynthesis started tissue specifically, with a high abundance in the endothelium of the integument in the ovules, followed by calreticulin accumulation in the embryo proper and in the associated endosperm at the late globular stage of embryogenesis. Using immunogold labeling, calreticulin was intracellularly localized with a high abundance to the Golgi compartment and to patches on the surface of dividing protoplasts. Smaller amounts were found in the endoplasmic reticulum and plasma membranes. The functional role of calreticulin in posttranslational processing and translocation processes, apart from its postulated function in cellular Ca2+ homeostasis, is discussed.

Amino Acid Sequence↗

PubMed1997

Physiological characterization of a microbial sensor containing the yeast Arxula adeninivorans LS3.

The yeast Arxula adeninivorans LS3 is a suitable organism for use as part of a microbial sensor. In combination with an amperometric oxygen electrode the sensor offered a possibility for the physiological characterization of this yeast. About 300-400 measurements could be carried out with a single Arxula sensor. The microbial sensor was remarkably stable for over 35 days, when kept at 37 degrees C during the operation time and at room temperature overnight. The physiological characteristics of Arxula adeninivorans LS3 obtained with the sensor technique were identical to the data obtained with the conventional techniques. However, the sensor technique makes it additionally possible to quantify the physiological data. So the substrates ribose, citric acid, glycerol, oil and benzoate produced signals lower than 10% in comparison to the glucose signal. Fructose, xylose, sucrose, maltose, gentianose, glucosamine, glutamic acid, tryptophan, butyric acid, lauryl acid and propionic acid reached 10-70%, galactose, alanine, glycine, lysine and methionine signals were similar to the glucose signal whereas acetic acid, ethyl alcohol, capron acid, capryl acid and caproic acid reached the highest signals up to 434%.

Amino Acids↗

PubMed1996

Classification of tyrosine kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains.

Two new kinases of Dictyostelium discoideum were identified by screening of a (lambda)gt11 expression library with a phosphotyrosine specific antibody. Amino-acid sequences derived from cDNA and genomic clones indicate that DPYK3 is a protein of 150 kDa and DPYK4, a protein of 75 kDa. The C-terminal fragments of each protein were produced in Escherichia coli and shown to be autocatalytically phosphorylated at tyrosine residues. A common feature of these kinases is the presence of two different sequence stretches in tandem that are related to kinase catalytic domains. The sequence relationships of DPYK3 and 4 to other protein kinases, and the positions of their catalytic domain sequences within the phylogenetic tree of protein kinases were analysed. Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two previously described tyrosine kinases of D. discoideum, a branch of their own, separate from the tyrosine kinase domains in sensu strictu. Domain II in DPYK4 is found on a different branch close to serine/threonine kinases.

Amino Acid Sequence↗

PubMed1996

Slow-speed freezing of chemically unfixed biological tissues and long-term storage of frozen samples for cryoscanning electron microscopy.

We describe a procedure in which plant tissue as well as a yeast culture on agar are frozen with slow cooling rates for observation of surface structures in a cryoscanning electron microscope. A system is also presented for long-term storage of frozen specimens under liquid nitrogen, in which the material is maintained for direct observation. Some small tools are described, which are essential for making preparations using slow-speed freezing and for the storage of prepared samples. Three examples of preparations with different complications are given: the "sculptures" on the surface of a leaf of Allium schoenoprasum, an early stage of flower development of Allium cernuum, and a part of an agar-grown colony of Arxula adeninivorans. In our experience, it is possible to store fully hydrated samples under the described conditions for more than a year without damaging the fine structures.

Allium↗

PubMed1996

The use of the single cell gel electrophoresis assay in detecting DNA single strand breaks in lung cells in vitro.

DNA single strand breaks (SSB) can be used as a biomarker of oxidant exposure, and also as an indicator of the carcinogenicity/ mutagenicity of a substance. The single cell gel electrophoresis (SCGE) assay is more sensitive and requires fewer cells compared to other techniques used for detecting SSB. We examined the utility of using the SCGE assay for human lung cells exposed to endogenous and exogenous oxidants. A human bronchial cell line (BEAS) was used as a model of airway epithelial cells in this study. BEAS cells exposed to 0-50 microM hydrogen peroxide (H2O2) for 60 min at 4 degrees C exhibited a concentration-dependent increase in SSB as determined by an increased DNA migration area in a gel undergoing electrophoresis. H2O2-induced increases in DNA SSB were also demonstrated using cultured normal human tracheobronchial epithelial (NHBE) cells and human alveolar macrophages in a concentration response manner. BEAS cells were also exposed to air or ozone (O3) on a Transwell filter without medium present apically. Cells exposed to O3 at 0.1 or 0.4 ppm at 37 degrees C for up 120 min had a time- and concentration-dependent increase in SSB compared to air-exposed cells. NHBE cells exposed to 0.4 ppm O3 (60 min) also had increased DNA SSB. Cells with H2O2-induced DNA SSB can be frozen and stored up to 4 weeks without altering the original DNA SSB. These findings indicate that SCGE can be used to detect SSB in cultured lung cells, and has applicability for detecting SSB in lung cells recovered from in vivo and in vitro exposures to oxidants.

Bronchi↗

PubMed1995

Temperature-dependent dimorphism of the yeast Arxula adeninivorans Ls3.

Arxula adeninivorans Ls3 is described as an ascomycetous, arthroconidial, anamorphic, xerotolerant yeast, which was selected from wood hydrolysates in Siberia. By using minimal salt medium or yeast-extract-peptone-medium with glucose or maltose as carbon source it was shown that this yeast is able to grow at up to 48 degrees C. Increasing temperatures induce changes in morphology from the yeast phase to mycelia depending on an altered programme of gene expression. This dimorphism is an environmentally conditioned (reversible) event and the mycelia can be induced at a cultivation temperature of 45 degrees C. Depending on the morphology of strain Ls3 (yeast phase or mycelia) the secretion behaviour as well as the spectrum of polypeptides accumulated in the culture medium changed. The activities of the accumulated extracellular enzymes glucoamylase and invertase were 2 to 3 times higher in cultures grown at 45 degrees C than in those grown at 30 degrees C. While the level of the glucoamylase protein secreted from mycelia between 45 and 70 hours did not change, biochemical activity decreased after a cultivation time of 43 hours. It was shown that this effect depended on both the catabolic repression of the glucoamylase by glucose and the thermal inactivation of this enzyme in media without or with low concentrations of starch or maltose.

Ascomycota↗

PubMed1995

Development of a two-site immuno-PCR assay for hepatitis B surface antigen.

Hepatitis B virus (HBV) gene transcription may occur at very low levels resulting in HBsAg concentrations in serum and liver below the limit of detection by currently available immunoassays. An assay has been developed that combines the specificity of two high affinity anti-HBs monoclonal antibodies (MAb) directed against distinct and separate determinants in the 'a' domain of HBsAg with the highly sensitive polymerase chain reaction (PCR) detection method. Following capture of HBsAg present in serum samples, the second anti-HBs MAb, which is biotinylated, is added. Binding of the second antibody allows the subsequent specific binding of streptavidin and a biotinylated linear DNA molecule derived from a bluetongue virus (BTV) gene. Presence of this DNA is then detected by PCR using BTV-specific primers. The PCR product is quantified by a liquid-phase oligonucleotide enzymatic assay, which further increases the sensitivity of the technique. The use of a two-site MAb capture and PCR detection system for HBsAg was shown to greatly enhance the specificity and sensitivity of the assay and detect as little as 0.5 pg of HBsAg in serum samples. It is suggested that the principles of this technique could be applied to measure other low level viral antigens in serum and biological samples.

Antibodies, Monoclonal↗

PubMed1993

Correlation of myosin heavy chain expression in the rat with cAMP in different models of hypertension-induced cardiac hypertrophy.

Both genetically determined and artificially-induced hypertension lead to cardiac hypertrophy and shift the myosin heavy chain (MHC) expression to the beta-MHC form. The cause of this change in gene expression is unknown. To contribute to the understanding of this phenomenon, we correlated the MHC expression in the left ventricle with basal, Forskolin- and isoprenaline-stimulated adenylate cyclase activity (cAMP production of membrane fractions). We used two control rat strains [Wistar-Hagemann (WH), Wistar-Kyoto (WKY)] and several rat models of hypertension: one clip-one kidney (1C-1K), desoxycorticosterone-treated rats (DOCA), rats with reduced renal mass (RRM) and spontaneously hypertensive rats (SHR). The level of hypertension correlated positively with the degree of cardiac hypertrophy (P < 0.01) and negatively (P < 0.05) with cAMP production, e.g. the higher the degree of hypertension, the lower both basal and stimulated cAMP levels. In addition we found that the lower the basal, isoprenaline- and Forskolin-stimulated cAMP production the lower was the expression of the alpha-MHC isoenzyme (P < 0.05). Thus, our data suggest that the decreased alpha-MHC expression upon hypertension-induced cardiac hypertrophy could be mediated via decreased adenylate cyclase activity and thus decreased intracellular cAMP production.

Adenylyl Cyclases↗

PubMed1992

Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer.

We investigated the expression of myosin subunits (myosin heavy chains) as well as light chains and the in vivo phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the adult male European hamster (Cricetus cricetus L.). Two myosin heavy chain isoenzymes could be detected under native and denaturing electrophoretic conditions having high (alpha-myosin heavy chain) and low (beta-myosin heavy chain) enzymatic activity. Enzymatic activity of alpha- and beta-myosin heavy chain revealed a different temperature dependency. When temperature increased ATPase activity of the alpha-myosin heavy chain isoenzyme increased relatively more than ATPase activity of the beta-myosin heavy chain isoenzyme. Summer animals expressed predominantly the beta-myosin heavy chain (79% of total myosin) while during hibernation the alpha-myosin heavy chain expression increased to 53% of total myosin. Winter-active hamsters kept at 22 degrees C and 12 h day/night rhythm showed the same myosin heavy chain isoenzyme pattern as summer-active animals. Two myosin light chain forms were expressed in the ventricle of all animal groups. The in vivo phosphorylation level of the phosphorylatable myosin light chain decreased from 45% in summer-active hamster to 23% during hibernation.

Animals↗

PubMed1992

Comparison of three nonradioisotopic polymerase chain reaction-based methods for detection of human immunodeficiency virus type 1.

Three nonradioisotopic polymerase chain reaction (PCR)-based detection techniques were evaluated for sensitivity and specificity in detecting human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells. The Roche prototype HIV-1 PCR assay, the Du Pont enzyme-linked oligonucleotide sandwich assay (ELOSA), and the Gen-Probe hybridization protection assay (HPA) were compared with a standard radioisotopic oligonucleotide solution hybridization (OSH) technique. A panel of 111 well-characterized clinical samples that included peripheral blood mononuclear cells from 48 healthy, low-risk, HIV-1 antibody-negative subjects, 24 antibody-positive subjects with stable CD4 counts of less than 200/mm3, and 39 antibody-positive subjects with stable CD4 counts of greater than 800/mm3 were studied. Each method demonstrated good specificity, ranging between 96 and 100%; those of the OSH and ELOSA (Du Pont) were 100%, those of the HPA (Gen-Probe) were 100% with one probe and 96% with the other probe, and that of the HIV-1 PCR assay (Roche) was 96%. Sensitivities ranged from 96 to 100% for the low-CD4-count group, with the OSH, the HIV-1 PCR assay (Roche), and the HPA (Gen-Probe) all attaining a sensitivity of 100%. For the high-CD4-count group, sensitivities ranged from 69 to 97%, with the OSH attaining a sensitivity of 97% and the HPA attaining sensitivities of 97% with one probe and 95% with the other probe. These data indicate that the nonradioisotopic techniques are sensitive and specific for the detection of HIV-1 proviral DNA in clinical samples.

Base Sequence↗

PubMed1991

Different legumin protein domains act as vacuolar targeting signals.

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.

Amino Acid Sequence↗

PubMed1991

The influence of cold stress on the myosin heavy chain expression of cardiac and smooth muscle in normotensive and spontaneously hypertensive female rats.

Cold exposure (6 weeks at 4 degrees C) of normotensive (Wistar-Kyoto) and stroke-prone spontaneously hypertensive female rats led to cardiac hypertrophy (in stroke-prone spontaneously hypertensive rats), increased the level of plasma thyroxine, and increased the alpha-myosin heavy chain expression in the left ventricle. In contrast, myosin heavy chain expression of both main mesenteric artery and uterus was not affected by cold stress and chronic hypertension, suggesting different regulation of myosin heavy chain expression in smooth and cardiac muscle in vivo.

Animals↗

PubMed1991

Primary care in New York State: report and recommendations of the associated medical schools of New York.

Medical education in New York is unique in the country in its scope and its diversity. It is important, as we go forward, that these strengths be neither eroded nor compromised. The AMS member institutions are making a collective commitment to work together to promote changes that will improve medical education for all students by providing them with enriched experience in primary care. Our major resource is faculty. To whatever degree medical schools can influence career choice, it is essential to this aim that the best possible people are placed in the settings in which primary care is taught. The schools will intensify their efforts to recruit and retain such faculty and, in whatever way is appropriate to each institution, provide them with the stature needed to emphasize the value which the school places on primary care. The schools will also work to provide exposure to primary care early in a student's academic career given anecdotal evidence, at least, that such early experience can influence subsequent specialty choice. Finally, the medical schools will assume greater responsibility for graduate medical education. If, with state support, ambulatory teaching sites are developed, the schools will make every effort to assure that they are staffed with high-quality faculty. Residents and students must see primary care practiced with total commitment to quality. It is hoped that, with state-initiated improvements in the practice environment, the ultimate outcome will be an increase in the number of our graduates selecting primary care disciplines for their practices and locating in areas in need of physicians.2+ Corporation, and the Greater New York Hospital Association. We are ready to work with others toward our common objectives, and we call on all of those who share these concerns to participate with us.

Career Choice↗

PubMed1990

[The reaction of the heart to radiation. II. Clinical studies].

In a review the cardiac attendant reactions in tumor irradiation in thoracal area are represented by means of papers and experiences of our own. It is shown that the heart reacts differently on ionizing rays and in special cases it can become a critical organ for radiotherapy. The diagnostic practice and the phenomenon of pericardiac and myocardiac reactions are described in detail. Complications on coronary vessel system are not frequent but because of their clinical consequence always they have to take into consideration. Cardiac radioreactions can be transient or harmless but rarely can lead to vitally threatening situations. Therefore it is referred to symptoms, diagnostic proving and possible or necessary therapeutic measures. Because significant curative therapeutic results can be attained by radiotherapy in thoracal area and the chances for curing increase for many patients, a slight risk is justifiable for side-effects on healthy adjoining tissue. On the other hand undesirable side reactions can influence the life-quality of the tumor cured patients. Therefore a radiotherapy in intrathoracal tumors with the aim of healing without complications has to take into consideration the sensibility of heart in addition to those of lung and spinal marrow. Beside permanent development of physical-technical suppositions these biological aspects have a substantial influence on optimizing irradiation in thoracal area. Finally it is tried to formulate really existing possibilities reducing a risk for cardiac side-effects and to refer to prospective interesting problems.

Heart↗

PubMed1990

[The reaction of the heart to radiation. I. State of the knowledge based on animal experimental studies].

The aim of curative tumor therapy is healing without complications. Therefore the avoidance of undesirable side-effects in healthy tissue becomes more important to such a degree as the lifespan can be prolonged by therapy: In this review the experiences to cardiac reactions by ionizing radiations are represented from experiments on animals. The former conception of a marked radioresistance of the heart has to be revised to the hitherto presented investigations. Acute inflammatory reactions and late, often progressive alterations can develop in the heart like in other normal tissue. These late-effects especially are reflected at pericardium, myocardium and vessel system (microvessel system and coronaries) and not rarely cause functional injuries of the tissue. Actual investigations give special attention to pathogenesis of acute radioreaction. It is supposed that their explanation gives a better understanding for the process of independence and progress and with that a possibility for prophylactic or therapeutic measures.

Animals↗