Structural and functional contributions to the G1 blocking action of the retinoblastoma protein. (the 1992 Gordon Hamilton Fairley Memorial Lecture).

The retinoblastoma gene product (RB) contributes to normal cell growth control. One of its functions is manifest as a block to exit from G1, which is carried out by an RB subspecies which is un- or underphosphorylated. After RB phosphorylation, a process which occurs towards the end of G1 in cycling cells, the block is lifted allowing a cell to enter S.

The effect of cocaine on the physiologic response to hemorrhagic shock.

Background: Bradycardia is thought to be an uncommon and abnormal response to acute blood loss. A review of trauma patients (n = 84) admitted during a 1-year period with a systolic blood pressure of less than 100 mm Hg revealed that 45% had relative bradycardia (heart rate < 100 beats/minute). Cocaine use was recorded more often in this group (76% versus 26%; p < 0.

Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of free E2F.

The transcription factor E2F activates the expression of multiple genes involved in cell proliferation, such as c-myc and the dihydrofolate reductase gene. Regulation of E2F involves its interactions with other cellular proteins, including the retinoblastoma protein (Rb), the Rb-related protein p107, cyclin A, and cdk2. We undertook a detailed analysis of E2F DNA-binding activities and their cell cycle behavior in primary human T cells.

Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein.

The cellular protein p107 shares many structural and biochemical features with the retinoblastoma gene product, pRB. We have isolated a full-length cDNA for human p107 and have used this clone to study the function of p107. We show that, like pRB, p107 is a potent inhibitor of E2F-mediated trans-activation, and overexpression of p107 can inhibit proliferation in certain cell types, arresting sensitive cells in G1.

The role of sigmoidoscopy in the management of gunshot wounds to the buttocks.

The charts of all patients (n = 70) admitted over 26 months after sustaining a gunshot wound to the buttocks were reviewed to assess the role of physical examination, routine radiologic studies, and sigmoidoscopy in the evaluation of these patients. There were 68 men and 2 women. Sixteen patients underwent sigmoidoscopy, which demonstrated a rectal injury in 7.

Functional interactions of the retinoblastoma protein with mammalian D-type cyclins.

The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G1 cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with Rb-binding DNA tumor virus oncoproteins. Coexpression of the three D-type cyclins with the cyclin-dependent kinase (cdk4) in insect cells generated Rb kinase activity.

Increasing antibiotic dose decreases polymicrobial infection after hemorrhagic shock.

Hemorrhagic shock has been shown to increase the susceptibility to infection despite the administration of conventionally accepted doses of antimicrobial drugs. The efficacy of increasing antibiotic dose in a model of mixed gram-negative infection, both with and without hemorrhagic shock, was examined. Shock was induced by bleeding rats to a mean arterial pressure of 45 millimeters of mercury for 45 minutes followed by resuscitation with shed blood and saline solution.

Immunosuppressive activity of [MeBm2t]1-, D-diaminobutyryl-8-, and D-diaminopropyl-8-cyclosporin analogues correlates with inhibition of calcineurin phosphatase activity.

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, has recently been identified as a common target for cyclophilin A-cyclosporin A and FK506 binding protein 12-FK506 complexes. This study has examined the structure activity relationships of cyclosporin A (CsA) and three functionally distinct analogues, [MeBm2t]1-CsA, D-diaminobutyryl-8-CsA (Dab8-CsA), and D-diaminopropyl-8-CsA (Dap8-CsA). Immunosuppressive potency in T cell activation models, NF kappa B activation, and IL-2 mRNA transcription has been compared with analogue affinity for cyclophilin A and inhibition of calcineurin phosphatase activity.

The effect of protein binding on the adherence of staphylococci to prosthetic vascular grafts.

Staphylococcus epidermidis is the major bacterium causing prosthetic vascular graft infections and the ability of some S. epidermidis strains to produce slime is associated with pathogenicity. This study investigated the effect of protein binding of staphylococci as a possible mechanism affecting bacterial adherence to vascular graft materials.

Management of the surgical patient with multiple system organ failure.

The management of the surgical patient with multiple system organ failure (MSOF) remains a formidable problem. Despite advances in critical care, mortality from MSOF remains virtually unchanged since the syndrome was characterized almost two decades ago. At the present time, there are no modalities that can actively reverse established organ failure, hence the treatment of these patients consists of metabolic and hemodynamic support until the process reverses itself or until death.

Continuous infusion of cefazolin is superior to intermittent dosing in decreasing infection after hemorrhagic shock.

Standard doses of antibiotic administered by intermittent infusions after hemorrhagic shock have decreased efficacy in combating infection. This study compared identical quantities of cefazolin administered after shock as intermittent doses or as continuous infusions in a subcutaneous abscess model. One hour after resuscitation from shock, rats were inoculated with 2 x 10(8) Staphylococcus aureus subcutaneously on the dorsum and divided into three groups: (1) control rats, which received no drug treatment; (2) rats in the intermittent group, which received cefazolin at either 30 or 60 mg/kg intraperitoneally, 30 minutes prior to inoculation, then every 8 hours for three doses, and (3) rats in the continuous infusion group, which received cefazolin at either 30 or 60 mg/kg intraperitoneally, 30 minutes prior to inoculation, followed by cefazolin, 90 or 180 mg/kg, intraperitoneally by continuous infusion more than 24 hours after inoculation.

A Saccharomyces cerevisiae RAD52 allele expressing a C-terminal truncation protein: activities and intragenic complementation of missense mutations.

A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth.

Specific enzymatic dephosphorylation of the retinoblastoma protein.

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W.

Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli.

Hemorrhagic shock inhibits lipopolysaccharide-induced myelopoiesis in both germ-free and conventional rats.

Background: Bacterial translocation has been implicated in the alteration of the immune response after shock and trauma. This study examined the effect of bacterial translocation on myelopoiesis after hemorrhagic shock in germ-free and conventional rats.

Methods: Awake, unrestrained germ-free and conventional rats were bled to a mean arterial pressure of 30 mm Hg until the animal required infusion of 10% of the shed blood.

The role of laparoscopy in abdominal trauma.

Thirty-nine hemodynamically stable trauma patients were evaluated prospectively by laparoscopy before planned celiotomy. Laparoscopy was performed using a forward-viewing laparoscope connected to two high-resolution video monitors. The mechanism of injury was blunt trauma in eight, stab wounds (SWs) in 16, and gunshot wounds (GSWs) in 15.

Acute stabilization of the cervical spine by halo/vest application facilitates evaluation and treatment of multiple trauma patients.

The management of acute cervical spine injuries has traditionally used bed-based skeletal traction until all non-neurologic injuries have been evaluated. This treatment method substantially hinders the ability to transport patients and to perform imaging studies and surgical procedures. In contrast, early application of a halo/vest apparatus provides immediate cervical stabilization and facilitates the diagnostic work-up and treatment of the patients with multiple injuries.

Charged surface residues of FKBP12 participate in formation of the FKBP12-FK506-calcineurin complex.

The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin.

Identification of a growth suppression domain within the retinoblastoma gene product.

To date, all naturally occurring retinoblastoma susceptibility gene (RB) mutations known to be compatible with stable protein expression map to the T/E1A and cellular protein-binding region (the "pocket" domain). This domain extends from residue 379 to 792. When full-length RB and certain truncated forms were synthesized in human RB -/- cells, we found that the minimal region necessary for overt growth suppression extended from residue 379 to 928.

Plasmid recombination in a rad52 mutant of Saccharomyces cerevisiae.

Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain.

The retinoblastoma-susceptibility gene product becomes phosphorylated in multiple stages during cell cycle entry and progression.

The retinoblastoma-susceptibility gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. We characterized RB phosphorylation after mitogenic stimulation of primary human T lymphocytes, initially arrested in the G0 state. RB is phosphorylated in at least three steps when T cells are driven into the cell cycle.

PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor.

A randomized prospective clinical trial to determine the efficacy of interferon-gamma in severely injured patients.

Many aspects of the normal immune response are depressed after severe injury. Reduced monocyte human leukocyte antigen-DR (HLA-DR) levels have closely correlated with the development of major infection. After a pilot study with recombinant interferon-gamma (rIFN-gamma) showed restoration of depressed HLA-DR levels after major injury, a multicenter, prospective, randomized, double-blind trial was conducted.

Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling.

The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin. Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical.