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Biomedical subjects

M R Holt

Publications and source records attributed to M R Holt.

13 recordsLinked to original sources
PubMed2002

Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells.

FLAP is a new method for localized photo-labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo-labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of beta-actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G-actin and the much slower dynamics of filamentous F-actin simultaneously in living cells.

Actins↗
PubMed2001

Cell motility: proline-rich proteins promote protrusions.

Many proline-rich proteins participate in delivering actin monomers to specific cellular locations where actin-rich membrane protrusions, such as ruffles, filopodia and microspikes, are formed. These protrusions are necessary for cell motility. Actin monomer is usually delivered to the site of polymerization in the form of profilactin - a complex of G-actin with a polyproline-binding protein, profilin. Here, we describe proline-rich proteins involved in regulating actin polymerization and classify them according to their role in recruiting profilin to the membrane.

Actins↗
PubMed1999

Integrin-mediated cell adhesion: the cytoskeletal connection.

Members of the integrin family of cell adhesion molecules play a pivotal role in the interaction between animal cells and the extracellular matrix. This article reviews the evidence (i) that the integrin beta-subunit cytoplasmic domain is important in the localization of integrins to focal adhesions, and for integrin-mediated cell adhesion/spreading; and (ii) that the integrin beta-subunit can be linked to F-actin via the actin-binding proteins talin, alpha-actinin and filamin. Talin has two or more actin-binding sites, and three binding sites for the cytoskeletal protein vinculin. Because vinculin can also bind F-actin, it may cross-link talin and actin, thereby stabilizing the interaction. In addition, vinculin contains a binding site for VASP (vasodilator-stimulated phospho-protein), a protein which may serve to recruit a profilin/G-actin complex to talin, which has actin-nucleating activity. Evidence that talin, vinculin and alpha-actinin are important in the assembly of focal adhesions, obtained using antisense technology and protein microinjection, is reviewed. To analyse the role of talin in focal adhesions, we have disrupted both copies of the talin gene in mouse embryonic stem (ES) cells. Undifferentiated talin (-/-) ES cell mutants are unable to assemble focal adhesions when plated on fibronectin, whereas vinculin (-/-) ES cells are able to do so. Finally, the role of small GTP-binding proteins in the assembly of focal adhesions is discussed, along with our recent studies using streptolysin-O-permeabilized Swiss 3T3 cells which suggest that the GTP-binding protein ADP-ribosylation factor-1 (ARF-1) is important in targeting the protein paxillin to focal adhesions.

Actins↗
PubMed1998

ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts.

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPgammaS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPgammaS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion-like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Delta17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPgammaS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Delta17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

3T3 Cells↗
PubMed1998

The focal adhesion phosphoprotein, VASP.

Vasodilator-stimulated phosphoprotein (VASP) is associated with focal adhesions and areas of dynamic membrane activity, where it is thought to have an important role in actin filament assembly and cell motility. VASP contains a central proline-rich sequence which recruits the G-actin binding protein profilin. Localization of VASP to the leading edge of a migrating cell can lead to local accumulation of profilin, which in turn can supply actin monomers to growing filament ends. VASP binds to the focal adhesion proteins vinculin and zyxin and this probably directs the phosphoprotein to focal adhesions and the leading edge of stimulated cells. VASP functions as a binding intermediate between profilin and focal adhesion proteins. Intracellular pathogens, including Listeria monocytogenes, have coat proteins which bind VASP. This is one way in which these pathogens use VASP, and other proteins from the host cell, to assemble the actin filaments they require to move around the cytoplasm of infected cells and enter neighbouring cells. Understanding the role of VASP and other proteins in cell and bacterial motility is likely to lead to development of new therapeutic strategies for diseases including atherosclerosis and tumour growth, and for limiting the spread of intracellular pathogens.

Actins↗
PubMed1996

The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin.

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actinfilament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-l (residues 839-850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

Actins↗
PubMed1994

A pilot syringe exchange program in Washington, DC.

The Washington, DC City Council authorized a pilot syringe exchange program to operate for only 60 days at a single drug abuse treatment facility in the District. Only adults on the waiting list for treatment were eligible (n = 467). Of the 33 who enrolled, median duration of drug injection was 18 years. Twenty-seven participants denied needle sharing. Of 209 needles distributed, 69% were returned. Low enrollment might have been due to restrictive entry criteria, inconvenient location, incorrect syringe size, and attitudes of treatment staff. For future efforts to have a public health impact, wider accessibility will be needed.

Adult↗
PubMed1992

Mechanism of delayed intracranial hypertension after cerebroventricular infusions in conscious rats.

Prior studies showed that cerebroventricular infusions of artificial cerebrospinal fluid, 8 microliter/min for 10 min, followed by a 10 min rest and a 24 h infusion of 0.5 microliters/min, raised cerebrospinal fluid pressure (CSFp) of conscious, unrestrained rats after about 2 h. Here, we report that the 10 min infusion alone evoked a delayed, prolonged rise in CSFp. Pressure during the infusion itself rose and recovered quickly, as is usually reported. Pressure/volume tests, used to calculate resistance to outflow (Ro) and compliance (C), revealed that infusions increased Ro and decreased C, after a delay (P less than 0.05). The rise in CSFp after infusion was blocked by pretreatment with acetazolamide + ouabain (P less than 0.05), but the delayed changes in Ro and C were unaffected. We suggest that the 10 min infusion of a sterile, balanced salt solution has a primary effect that increases Ro; as CSF synthesis continues, C is exhausted and the delayed rise in CSFp ensues. This non-traumatic method of raising CSFp may be a useful method to study intracranial fluid dynamics.

Acetazolamide↗
PubMed1992

Drive-up prescription refill service at a large Navy medical facility.

A drive-up prescription refill service at a large naval medical facility is described. A pharmacy drive-up refill service was created to reduce customer congestion, to reduce demand for parking, and to improve customer service. The drive-up program is staffed by a full-time pharmacy technician, a full-time volunteer, and a part-time pharmacy technician who assists during lunches, breaks, and peak hours. Customers must request refills by telephone, and requests are recorded by a pharmacy answering machine. Recorded requests are transcribed, processed, and checked by a pharmacist in the main pharmacy. Refills ready for pickup are transported to the drive-up site twice daily. If a refill is missing, the customer is asked to park, fill out a missing refill form, and pick up the refill at the main pharmacy. Approximately 700 prescription refills are processed and filled daily at the drive-up service. The addition of a drive-up refill service reduced customer visits to the outpatient pharmacy department by about one third and reduced demand for parking by a projected 360-400 parking spaces per day. The error rate for missing refills is less than 0.5%. The only negative consequence of the drive-up service is less interaction between the customer and the pharmacy staff for counseling. A drive-up refill service at a large naval medical facility reduced customer congestion, reduced parking demand, and improved customer service at the outpatient pharmacy department.

Automobile Driving↗
PubMed1987

Pharmacy practice in the United States Navy.

The current status of pharmaceutical services in the United States Navy Medical Department is described. The mission of the Navy Medical Department is to provide comprehensive health care to the more than 2.7 million active-duty and retired Navy personnel and their dependents. A total of 144 Navy pharmacy officers, 50 civilian pharmacists employed by the Navy, and 650 Navy-trained technicians practice in 32 Naval hospitals. All Naval hospitals have unit dose drug distribution systems and complete i.v. admixture services as well as an extensive series of ambulatory-care clinics. Clinical pharmaceutical services provided in these facilities vary depending upon the size and purpose of the facility. Pharmaceutical services are supported by the TRIPHARM computer system. Navy pharmacy officers may choose to pursue full-time graduate studies or may follow career paths outside of pharmacy. Navy pharmacists currently serve in a variety of administrative positions, as instructors and curriculum directors in the two Navy technician training schools, and as commanding officers of drug screening laboratories. In times of combat, Navy pharmacists may be assigned to hospital ships or to medical facilities near the line of combat. The ability of Navy pharmacists to respond to innovations in pharmacy practice will be the key to the future success of Navy pharmacy practice. One of the major challenges facing Navy pharmacy in the next few years is the recruitment of qualified pharmacy officers.

Ambulatory Care↗
PubMedDate not supplied

The pharmacist's role in reducing patient delay in seeking treatment for acute myocardial infarction.

The most common reason for delay in treatment of an AMI is the patient's failure to seek care promptly. Individuals diagnosed with CHD, including those who have experienced an AMI, are considered to be at high risk for an AMI. These patients have the same or greater delay times as individuals without prior AMI or CHD. Pharmacists interact with these high-risk individuals and their families frequently in person or by telephone. During these interventions, they have the opportunity, through education and counseling, to improve their patients' understanding of early symptoms of AMI and the need for and benefits of prompt evaluation and treatment. Hearing this message from their pharmacist and from other health care providers in other settings will hopefully lead the high-risk individual to seek care promptly when needed. Successfully conveying this message could effectively reduce the morbidity and mortality associated with CHD.

Counseling↗