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Biomedical subjects

P K Herman

Publications and source records attributed to P K Herman.

18 recordsLinked to original sources
PubMed1997

Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.

Culture Media↗
PubMed1995

Restructuring. Implications for nursing administration education.

The Council on Graduate Education for Administration in Nursing (CGEAN) was established to further develop and improve graduate education for administration in nursing. The Council seeks to identify the nature and direction of education for administration in nursing in various healthcare systems, providing guidelines for programs offering administration. A major goal of CGEAN is facilitating dialogue between nursing service administrators and graduate level educators who are engaged in teaching and research related to administration in nursing. This column, sponsored by the members of the Council, analyzes and responds to position statements and trends related to the delivery of health services and graduate education for administrators in nursing.

Delivery of Health Care↗
PubMed1993

Correlation of myelin basic protein-like material in cerebrospinal fluid of multiple sclerosis patients with their response to glucocorticoid treatment.

Predictors and laboratory correlates of the response of patients with multiple sclerosis to glucocorticoids are not well defined. Our study was undertaken to determine if the levels of myelin basic protein (MBP)-like material in cerebrospinal fluid (CSF) might indicate which patients with multiple sclerosis would show a short-term (5 day) or intermediate-term (40 day) improvement of at least a full-grade Kurtzke disability score after initiating treatment with glucocorticoids. A total of 62 patients received 71 courses of treatment consisting of 5 days of intravenous methylprednisolone (500 mg per day) usually followed by a 4-week tapering dose of oral prednisone. CSF was obtained before initiation of treatment and analyzed for MBP-like material by radioimmunoassay. Results were analyzed by chi 2 tests of association and by logistic regression. Individuals having a CSF MBP-like material level of > or = 0.1 ng/ml overall showed a greater likelihood of continued improvement at day 40 (p = 0.014) or further improvement between days 5 and 40 (p = 0.003). Those in the first 15 days of worsening and with an elevated CSF MBP-like level were more likely to respond by day 5. Relapsing-remitting and relapsing-progressive forms of the disease were more likely to respond at both time points than were patients with primary or secondary chronic progressive patterns. The Kurtzke disability score at entry and the major anatomical site of the central nervous system symptomatically affected were not predictive of outcome at either time.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole.

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) are required for the sorting of soluble hydrolases to the yeast vacuole. Over-production of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vps15 kinase domain mutants, suggesting that Vps15p and Vps34p functionally interact. Subcellular fractionation and sucrose density gradients indicate that Vps15p is responsible for the association of Vps34p with an intracellular membrane fraction. Chemical cross-linking and native immunoprecipitation experiments demonstrate that Vps15p and Vps34p interact as components of a hetero-oligomeric protein complex. In addition, we show that an intact Vps15 protein kinase domain is required for activation of the Vps34 PI 3-kinase, suggesting that the Vps34 lipid kinase is regulated by a Vps15p-mediated protein phosphorylation event. We propose that Vps15p and Vps34p function together as components of a membrane-associated signal transduction complex that regulates intracellular protein trafficking decisions through protein and lipid phosphorylation events.

Biological Transport↗
PubMed1992

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.

Adult↗
PubMed1991

A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole.

The VPS15 gene encodes a novel protein kinase homolog that is essential for the efficient delivery of soluble hydrolases to the yeast vacuole. Point mutations altering highly conserved residues within the Vps15p kinase domain result in the secretion of multiple vacuolar proteases. In addition, the in vivo phosphorylation of Vps15p is defective in these kinase domain mutants, suggesting that Vps15p may regulate specific protein phosphorylation reactions required for protein sorting to the yeast vacuole. Subcellular fractionation studies further demonstrate that the 1455 amino acid Vps15p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. This association may be mediated by myristate as Vps15p contains a consensus signal for N-terminal myristoylation. We propose that protein phosphorylation may act as a molecular "switch" within intracellular protein sorting pathways by actively diverting proteins from a default transit pathway (e.g., secretion) to an alternative pathway (e.g., to the vacuole).

Amino Acid Sequence↗
PubMed1991

A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery.

The yeast VPS15 gene encodes a novel protein kinase homolog that is required for the sorting of soluble hydrolases to the yeast vacuole. In this study, we extend our previous mutational analysis of the VPS15 gene and show that alterations of specific Gps15p residues, that are highly conserved among all protein kinase molecules, result in the biological inactivation of Vps15p. Furthermore, we demonstrate here that short C-terminal deletions of Vps15p result in a temperature-conditional defect in vacuolar protein sorting. Immediately following the temperature shift, soluble vacuolar hydrolases, such as carboxypeptidase Y and proteinase A, accumulate as Golgi-modified precursors within a saturable intracellular compartment distinct from the vacuole. This vacuolar protein sorting block is efficiently reversed when mutant cells are shifted back to the permissive temperature; the accumulated precursors are rapidly processed to their mature forms indicating that they have been delivered to the vacuole. This rapid and efficient reversal suggests that the accumulated vacuolar protein precursors were present within a normal transport intermediate in the vacuolar protein sorting pathway. In addition, this protein delivery block shows specificity for soluble vacuolar enzymes as the membrane protein, alkaline phosphatase, is efficiently delivered to the vacuole at the non-permissive temperature. Interestingly, the C-terminal Vps15p truncations are not phosphorylated in vivo suggesting that the phosphorylation of Vps15p may be critical for its biological activity at elevated temperatures. The rapid onset and high degree of specificity of the vacuolar protein delivery block in these mutants suggests that the primary role of Vps15p is to regulate the sorting of soluble hydrolases to the yeast vacuolar compartment.

Fungal Proteins↗
PubMed1991

Cerebrospinal fluid levels of myelin basic protein-like material and soluble interleukin-2 receptor in multiple sclerosis.

The presence, level and disease activity relationships of soluble interleukin-2 receptor (sIL-2R) in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients are unresolved. We measured CSF immunoreactive myelin basic protein (MBP), a marker of acute myelin damage, and sIL-2R levels in the CSF from 11 patients with active relapsing remitting (RR) MS, five with stable RR MS, eight with chronic progressive (CP) MS, five with other neurologic diseases, and three normal controls. No measurable (less than 100 units/ml) sIL-2R was present in any of the samples. Conversely, MBP levels were elevated in the active RR group compared to the other four groups. These results indicate that, at the sensitivity of assays currently available, levels of CSF sIL-2R do not correlate with the diagnosis or disease activity of MS.

Humans↗
PubMed1991

Changes induced in astrocyte cathepsin D by cytokines and leupeptin.

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were greater than 95% astrocytes and less than 3% microglia. Under serum-free conditions, leupeptin induced a 1.4- to 2.0-fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon-gamma, human recombinant tumor necrosis factor-alpha, human recombinant interleukin-1 beta, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.

Analysis of Variance↗
PubMed1990

Conformational correlates of the epitopes of human myelin basic protein peptide 80-89.

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.

Circular Dichroism↗
PubMed1990

Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

Amino Acid Sequence↗
PubMed1990

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.

Alleles↗
PubMed1990

The fungal vacuole: composition, function, and biogenesis.

The fungal vacuole is an extremely complex organelle that is involved in a wide variety of functions. The vacuole not only carries out degradative processes, the role most often ascribed to it, but also is the primary storage site for certain small molecules and biosynthetic precursors such as basic amino acids and polyphosphate, plays a role in osmoregulation, and is involved in the precise homeostatic regulation of cytosolic ion and basic amino acid concentration and intracellular pH. These many functions necessitate an intricate interaction between the vacuole and the rest of the cell; the vacuole is part of both the secretory and endocytic pathways and is also directly accessible from the cytosol. Because of the various roles and properties of the vacuole, it has been possible to isolate mutants which are defective in various vacuolar functions including the storage and uptake of metabolites, regulation of pH, sorting and processing of vacuolar proteins, and vacuole biogenesis. These mutants show a remarkable degree of genetic overlap, suggesting that these functions are not individual, discrete properties of the vacuole but, rather, are closely interrelated.

Fungi↗
PubMed1988

Human myelin basic protein peptide 69-89: immunochemical features and use in immunoassays of cerebrospinal fluid.

The characteristics and heterogeneity of the myelin basic protein (MBP)-like material appearing in cerebrospinal fluid (CSF) after acute central nervous system (CNS) myelin injury are unresolved. Antigenic material containing an epitope in the carboxyl-terminal portion of human MBP peptide 45-89 (from the intact molecule of 170 residues) is a prominent species of the MBP-like material present. In an effort to define further the MBP-like material in CSF and to enhance its detection, a modified double-antibody radioimmunoassay has been developed using a radioligand of human MBP synthetic peptide 69-89. This assay is more sensitive with results paralleling those of previously used MBP assays for CNS myelin damage. Results with this assay provide additional confirmation of the presence of an epitope of MBP in the decapeptide of MBP 80-89 but in a conformation simulating that of intact MBP in CSF after CNS myelin injury. Unexpected buffer effects were noted to influence the immunochemical behavior of some of the small peptides of MBP.

Binding, Competitive↗
PubMed1988

Myelin basic protein and magnetic resonance imaging for diagnosing radiation myelopathy.

The identification of radiation myelopathy using biochemical assays and imaging techniques has not previously been accomplished but has clear clinical application. Measurement of myelin basic protein (MBP) in the cerebrospinal fluid (CSF) and visualization of the spinal cord using magnetic resonance imaging (MRI) gives a potentially accurate diagnosis of radiation myelopathy. Female New Zealand white rabbits were irradiated to the thoracic spinal cord with single doses of 15-45 Gy. Animals receiving higher doses (greater than or equal to 22 Gy) generally demonstrated an early paresis (4-8 weeks) that temporarily improved, and then progressed to complete paralysis by 14-18 weeks. MBP levels in the CSF became strikingly elevated to 100-1000 times the normal value. Subsequent, experiments in which rabbits were serially assessed for MBP levels demonstrated a transient elevation, which corresponded to the transient paresis, followed by dramatic elevations concurrent with the onset of paralysis. Magnetic resonance imaging (MRI) of the irradiated spinal cord showed a geographically distinct region of abnormality that corresponded to the radiation field. Histopathology demonstrated demyelination, focal astrocytosis, erythrodiapedesis, and perineuronal edema in the irradiated sections. It appears that MBP levels in the CSF reflect not only radiation-induced myelopathy but also transient demyelination, and that MRI may have the potential to indicate the region of damage.

Animals↗
PubMed1987

Myelin basic protein-specific RNA levels in interleukin-2-stimulated oligodendrocytes.

In the immune system, T- and B-cell proliferation, as well as B-cell immunoglobulin secretion, is induced by interleukin-2 (IL-2), a T-cell-derived lymphokine. IL-2 also influences the growth of glial cells, specifically, the proliferation and maturation of oligodendrocytes. Studies were conducted to investigate further IL-2-induced maturation of oligodendrocytes through its effect on the regulation of the myelin basic protein (MBP) gene. A cDNA probe specific for rat MBP and a double-antibody radioimmunoassay for MBP were used to quantitate MBP mRNA and protein levels in oligodendrocytes under different experimental conditions. We demonstrate that both MBP mRNA and protein levels are increased in IL-2-stimulated oligodendrocytes. MBP mRNA levels increase within 8 h after IL-2 stimulation, peak between 24 and 48 h, and then decline slightly. MBP protein levels increase 24 h after stimulation and peak at 72 h. MBP mRNA transcripts in the range of 2.0-2.4 kilobases are present in cultured rat oligodendrocytes, which are similar to the MBP mRNA transcripts detected in whole rat and mouse brain. These mRNA transcripts are specifically increased in quantity after oligodendrocyte stimulation with IL-2. These results suggest that one component of oligodendrocyte differentiation/maturation--MBP mRNA and protein expression--can be regulated in part by IL-2.

Animals↗