Failure to fail: the institutional perspective.

PURPOSE: To determine institutional barriers to placing failing students on probation, dismissing students. METHODS: An online survey study was distributed to Student Affairs Deans or the equivalent at allopathic (MD) and osteopathic (DO) medical schools, and physician assistant (PA) and nurse practitioner (NP) schools across the United States. Nineteen (40%) of the 48 schools responded: six MD, four DO, five PA and four NP. The survey contained demographic questions and questions regarding probation and dismissal. Themes were independently coded and combined via consensus based on grounded theory. The survey was distributed until saturation of qualitative responses were achieved. RESULTS: Respondents identified variations in the use of probation and dismissal and a wide range of barriers, with the greatest emphasis on legal concerns. Respondents felt that students were graduating who should not be allowed to graduate, and that the likelihood of a student being placed on probation or being terminated was highly variable. DISCUSSION: Our results suggest that institution culture at heath professions schools across the United States may represent an obstacle in placing failing learners on probation and dismissing learners who should not graduate. Additional studies are needed to prove if these concerns are founded or merely fears.

Fyn kinase-tubulin interaction during meiosis of rat eggs.

Prior to fertilization, the spindle of vertebrate eggs must remain stable and well organized during the second meiotic meta-phase arrest (MII). In a previous study we have determined that the completion of meiosis is a Src family kinase (SFK)-dependent event. In the current study we have used the SFK inhibitors, SU6656 and PP2, and demonstrated that inhibition of SFKs caused the formation of a disorganized spindle. The observation that proper organization of an MII spindle is an SFK-dependent process, combined with our previous finding that Fyn kinase is localized at the microtubules (MTs), prompted us to examine the potential role of Fyn in MT signaling. Our results show an association between Fyn and tubulin, the ability of Fyn to phosphorylate tubulin in vitro and stimulation of meiosis completion by injection of a constitutively active form of Fyn (CAF). We suggested that SFKs mediate significant functions during the organization of the MII spindle. In view of CAF injection experiments, and of the pronounced concentration of Fyn kinase at the spindle, we propose that Fyn may play an important role in some aspects of the spindle functions, possibly those involving the MTs.

Fertilization of sea urchin eggs and sperm motility are negatively impacted under low hypergravitational forces significant to space flight.

Sperm and other flagellates swim faster in microgravity (microG) than in 1 G, raising the question of whether fertilization is altered under conditions of space travel. Such alterations have implications for reproduction of plant and animal food and for long-term space habitation by man. We previously demonstrated that microG accelerates protein phosphorylation during initiation of sperm motility but delays the sperm response to the egg chemotactic factor, speract. Thus sperm are sensitive to changes in gravitational force. New experiments using the NiZeMi centrifugal microscope examined whether low hypergravity (hyperG) causes effects opposite to microG on sperm motility, signal transduction, and fertilization. Sperm % motility and straight-line velocity were significantly inhibited by as little as 1.3 G. The phosphorylation states of FP130, an axonemal phosphoprotein, and FP160, a cAMP-dependent salt-extractable flagellar protein, both coupled to motility activation, showed a more rapid decline in hyperG. Most critically, hyperG caused an approximately 50% reduction in both the rate of sperm-egg binding and fertilization. The similar extent of inhibition of both fertilization parameters in hyperG suggests that the primary effect is on sperm rather than eggs. These results not only support our earlier microG data demonstrating that sperm are sensitive to small changes in gravitational forces but more importantly now show that this sensitivity affects the ability of sperm to fertilize eggs. Thus, more detailed studies on the impact of space flight on development should include studies of sperm function and fertilization.

Transient nuclear localization of Fyn kinase during development in zebrafish.

Fyn protein tyrosine kinase is present in the unfertilized and fertilized egg, becomes activated within minutes following fertilization, and has been localized to the cortical cytoplasm and spindle apparatus of the zygote. In order to establish the expression pattern of Fyn in the early embryo, we examined the distribution pattern of Fyn by immunofluorescence microscopy. Fyn protein is distributed evenly among cells of the cleavage stage zebrafish embryo and is concentrated in the cortical region of each cell. During blastula and gastrula stages, Fyn was expressed in all cells, however a subpopulation of cells exhibited strong nuclear staining for Fyn. Nuclear Fyn staining was not observed after the gastrula period of development, nor in the adult zebrafish. Immunoprecipitation of Fyn from isolated mid-blastula nuclei confirmed Fyn was present in the nucleus. This is, to our knowledge, the first demonstration of Fyn kinase, which lacks a nuclear localization signal, present in the nucleus. The transient compartmentalization of Fyn in the nucleus could be important in nuclear signaling.

Role of the Fyn kinase in calcium release during fertilization of the sea urchin egg.

Protein tyrosine kinase activity has been implicated as part of the signaling mechanism leading to the sperm-induced calcium transient following fertilization. In the present study, we have tested the role of the Fyn kinase in triggering the calcium transient by microinjecting domain-specific fusion proteins encoding regions of Fyn sequence as inhibitors of Fyn function in vivo. A fusion protein encoding the SH2 domain of Fyn caused an increase in the latent period between sperm-egg fusion and the beginning of the calcium transient and reduced the amplitude of the calcium signal. A fusion protein encoding the U + SH3 domains also caused a small increase in the latent period. Microscopic examination revealed that a large percentage of eggs injected with the U+SH3 or SH2 domains became polyspermic as a result of the delayed block to polyspermy. Affinity experiments demonstrated that the U+SH3 and SH2 domains of Fyn were capable of forming a stable complex with phospholipase Cgamma from the sea urchin egg. The results suggest that the Fyn kinase participates in the signaling events leading up to the calcium transient and may directly regulate phospholipase Cgamma activity at fertilization.

Addiction: part II. Identification and management of the drug-seeking patient.

The medications most often implicated in prescription drug abuse are opioid analgesics, sedative-hypnotics and stimulants. Patients with acute or chronic pain, anxiety disorders and attention-deficit disorder are at increased risk of addiction comorbidity. It is important to ask patients about their substance-use history, including alcohol, illicit drugs and prescription drugs. Patients who abuse prescription drugs may exhibit certain patterns, such as escalating use, drug-seeking behavior and doctor shopping. A basic clinical survival skill in situations in which patients exert pressure on the physician to obtain a prescription drug is to say "no" and stick with it. Physicians who overprescribe can be characterized by the four "Ds"-dated, duped, dishonest and disabled. Maintaining a current knowledge base, documenting the decisions that guide the treatment process and seeking consultation are important risk-management strategies that improve clinical care and outcomes.

Fertilization triggers activation of Fyn kinase in the zebrafish egg.

Fertilization results in the tyrosine phosphorylation of several egg proteins and studies have shown that tyrosine protein kinase activity is required for successful fertilization. The Fyn protein kinase has been detected in eggs of the sea urchin, frog and rat, although measurement of fertilization-induced changes in Fyn kinase activity have only been successful in the sea urchin system. The present study demonstrates the presence of Fyn kinase in the zebrafish egg and the stimulation of this enzyme at fertilization. Activation of Fyn was detected as early as 30 seconds post-fertilization and increased approximately six-fold by 2 minutes post-insemination. The activation of Fyn in the zebrafish egg required sperm and was not observed in spontaneously activated eggs.

Characterization of human papillomavirus in airway papillomas by histologic and biochemical analysis.

The role of human papillomavirus (HPV) in airway papillomas has been well defined in recent literature. The chronicity and recurrence of papillomas has been postulated to be a result of residual viral genome in tissue treated with standard laser techniques. Thirteen patients with airway papillomas were selected for study with polymerase chain reaction (PCR) methods to detect viral DNA. Specimens taken prior to laser therapy and specimens taken at laser margins were consistently positive for HPV DNA by PCR. The HPV DNA is apparently present in tissues after macroscopic disease has been ablated by laser techniques. Histologic analysis of laser biopsies demonstrated fragments of squamous epithelium with cytologic features of HPV infection. Laser treatment is ineffective in eradicating HPV-infected tissues from airway papillomas, and this finding supports the notion that recurrence is a product of HPV incorporated into tissue not ablated by laser irradiation. Specific methods, results, and clinical correlation will be discussed.

Fertilization-induced activation of phospholipase C in the sea urchin egg.

Fertilization results in the biphasic activation of polyphosphoinositide-specific phospholipase C (PLC) activity with an initial increase in activity coincident with the sperm-induced calcium transient, followed by a more sustained increase prior to mitosis. Immunoprecipitation studies demonstrated that the gamma isoform of PLC is present in both the unfertilized and the fertilized egg and contributes to the initial phase of PLC activation. Fertilization also resulted in translocation of a significant fraction of PLC-gamma from the cytosol to the membrane compartment of the egg.

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg.

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.

Expression and immunolocalization of p59c-fyn tyrosine kinase in rat eggs.

Fertilization overcomes meiotic arrest by triggering a series of biochemical events, resulting in activation of the egg. A small group of protein tyrosine kinases (PTKs) have been identified in eggs of invertebrates and lower vertebrates and inhibitor studies have suggested that they play a role in late events of egg activation. A recent study using the sea urchin system demonstrated that Fyn kinase was expressed in eggs and was activated within minutes of fertilization. In the present study, Western blot analysis as well as immune complex kinase assay demonstrated that p59(c-fyn) kinase was expressed in both unfertilized and fertilized rat eggs. Immunofluorescence confocal microscopy demonstrated that Fyn kinase was localized to the egg cortex but also to the polar body and the fertilizing cone which are elevated from the cortical cytoplasm of the activated egg. Surprisingly, Fyn was also found to be highly concentrated over the meiotic and mitotic spindles. To date, Fyn is the first PTK demonstrated to be present in the mammalian egg. Localization of Fyn to the egg cortex as well as the spindle microtubules indicates that this protein kinase may have multiple functions within the egg.

Protein tyrosine kinase activity following fertilization is required to complete gastrulation, but not for initial differentiation of endoderm and mesoderm in the sea urchin embryo.

The egg activation process functions to implement developmental programs that act much later in embryogenesis. One example of this is the fact that application of protein tyrosine kinase inhibitors to the fertilized sea urchin egg for a 15-min period results in a defect in the gastrulation process occurring over 24 h later (Kinsey, W. H., Dev. Biol. 172, 704-707, 1995). In the present study, we show that the window of sensitivity is not due to differential uptake of inhibitor, and establish that the inhibitor inhibits tyrosine kinase activity at the time of application. We also demonstrate that inhibition of protein tyrosine kinase activity in the zygote causes a specific defect in the morphogenetic movements associated with gastrulation without interfering with the initial specification and differentiation of endoderm and mesoderm. Differentiation events occurring concurrent with or subsequent to gastrulation were also suppressed in embryos derived from treated zygotes. These findings indicate that fertilization initiates a signaling cascade involving protein tyrosine kinase activity that is required specifically for events at gastrulation. This signaling event is required to complete the developmental program of both endoderm and mesoderm, but is different from those events necessary for initial specification of endodermal and mesodermal cell fate.

Tyrosine kinase signaling at fertilization.

The unfertilized egg is a highly differentiated cell that retains unlimited developmental potential. The execution of that potential requires signal transduction pathways that release the egg from its quiescent metabolic state, direct the union of the maternal and paternal genome, and initiate a developmental program that will guide embryogenesis. The egg is equipped with an array of cytosolic as well as cell surface receptor protein tyrosine kinases as part of a preassembled signal transduction mechanism. These protein tyrosine kinases have been found to act at several points during this egg activation process, beginning as early as the initial sperm-egg interaction. While many of these kinase functions are common to all cells, several functions unique to fertilization demonstrate the versatility of this class of protein kinases.

Fertilization promotes selective association of the Abl [correction of AbI] kinase with the egg cytoskeleton.

Fertilization of the sea urchin egg is known to involve activation of a variety of protein kinases including at least one protein tyrosine kinase. In the present study, fertilization was found to stimulate the association of the Abl [correction of AbI] protein kinase with the detergent-insoluble egg cytoskeleton. Increased levels of Abl protein and of protein tyrosine kinase activity were detected in cytoskeleton preparations made as early as 5 to 15 min after insemination. Immunofluorescence localization demonstrated that the Abl kinase becomes associated primarily with the cortical cytoskeleton of the fertilized egg. A separate, 57 kDa protein tyrosine kinase did not associate with the cytoskeleton indicating that fertilization results in the selective association of the Abl kinase with the cortical cytoskeleton.

Biphasic activation of Fyn kinase upon fertilization of the sea urchin egg.

Fertilization results in the tyrosine phosphorylation of several egg proteins within minutes of sperm-egg binding and inhibitor studies have shown that tyrosine kinase activity is required for many aspects of egg activation. The present study demonstrates the presence of p59c-fyn kinase in the sea urchin egg and examines the effect of fertilization on the activity of this enzyme. Fertilization had little effect on Fyn kinase activity during the first 2 min after insemination; however, activity had increased approximately eightfold between 5 and 15 min postinsemination. This initial, rapid increase in kinase activity was followed by a period of slightly elevated kinase activity, which was two- to threefold higher than that in the unfertilized egg. Bindin, as well as various parthenogenic agents known to activate the calcium- and pH-mediated pathways of egg activation, failed to elicit any change in enzyme activity, indicating that activation of the kinase required sperm-induced egg activation. However, phorbol ester treatment did induce a slow increase in kinase activity within 30 to 60 min of administration. These findings indicate that the p59fyn kinase is activated within minutes of fertilization and may play a role in the egg activation process.

Protein tyrosine kinase activity during egg activation is important for morphogenesis at gastrulation in the sea urchin embryo.

Fertilization triggers a series of preprogrammed events functioning to activate egg metabolism, incorporate the paternal genome, and initiate development. The activity of protein tyrosine kinases during egg activation is required for several steps leading to the first cell division. We now present evidence for an additional protein tyrosine kinase-mediated event that occurs between 30 and 45 min after insemination and is not required until gastrulation, which occurs over 24 hr later. Eggs treated with protein tyrosine kinase inhibitors within this window of time cleaved and formed normal blastulae but could not gastrulate or undergo further development to the pluteus stage. These findings provide the first evidence that some of the control mechanisms used in later development are established during a brief period of time in the fertilized egg and require the action of one or more protein tyrosine kinases.

Effects of protein tyrosine kinase inhibitors on egg activation and fertilization-dependent protein tyrosine kinase activity.

Fertilization results in the activation of protein tyrosine kinases within minutes of sperm-egg binding, although the role of the kinase(s) involved is not clear. In the present study, we have treated sea urchin eggs with genistein, as well as other protein tyrosine kinase inhibitors, and have characterized the subsequent effect on fertilization and egg activation. Genistein treatment of sea urchin eggs inhibits the overall fertilization-dependent tyrosine kinase activity as well as the specific phosphorylation of a 350-kDa protein, but it did not inhibit cAMP-dependent kinase and had little effect on protein kinase C at concentrations less than 100 microM. Genistein, erbstatin, and tyrphostin B42 did not inhibit the early events of fertilization such as elevation of the fertilization envelope; however, later events such as pronuclear migration, DNA synthesis, and cell division were inhibited. These results suggest that protein tyrosine kinases activated following fertilization play a role in the later events of egg activation such as the initiation of pronuclear movement and entry into the S phase of the cell cycle.

Differential phosphorylation of a 57-KDa protein tyrosine kinase during egg activation.

Fertilization results in activation of many protein kinases which function during egg activation. We have used metabolic labelling and immunoprecipitation to study changes in the phosphorylation state of a 57-KDa src-family protein tyrosine kinase during fertilization of the sea urchin egg. The kinase was phosphorylated on serine at all periods studied but it was also phosphorylated transiently on tyrosine at 5 minutes post insemination and then on threonine at 90 minutes after fertilization. These data indicate that the 57-KDa PTK may be under complex regulatory control during the first cell cycle.

Identification of an abl-related protein tyrosine kinase in the cortex of the sea urchin egg: possible role at fertilization.

Fertilization results in the tyrosine phosphorylation of several egg proteins within minutes of sperm-egg binding, although the identity of the kinase(s) involved and the mechanism of regulation is not known. In the present study, we have used site-directed antibodies based on the predicted amino acid sequence of a sea urchin egg transcript that shares significant homology with members of the ABL family of protein tyrosine kinases. These antibodies identified a 220-kDa protein kinase, highly enriched in the egg cortex, where it is tightly associated with detergent-insoluble cytoskeletal elements. The enzyme is capable of phosphorylating synthetic peptide substrates which were used to characterize the kinase activity in an immune-complex assay. Measurement of the protein tyrosine kinase activity immunoprecipitated at different times after fertilization revealed that the level of kinase activity is transiently elevated during the first few minutes postinsemination. Western blot analysis indicated that the amount of the 220-kDa protein did not increase significantly during this period, so the increased kinase activity probably results from activation of the enzyme. These in vitro studies indicate that the 220-kDa abl-related kinase is one of the protein kinases activated during fertilization and suggest that it may play a role in the egg activation process.