Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches.

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 microM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase.

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

CT detection of occult pneumothorax in multiple trauma patients.

Chest injuries are the cause of death in 25% of trauma fatalities, and a major contributing factor in an additional 50%. Pneumothorax, the second most common chest injury, may often be initially overlooked. Administration of anesthesia and mechanical ventilation may produce enlargement of a pneumothorax and clinical deterioration. We reviewed 90 trauma patients who had been admitted with a diagnosis of pneumothorax or who had developed pneumothoraces after hospital admission. In 35 cases (38.8%), initial supine chest x-ray study failed to detect a pneumothorax, and the diagnosis was made on CT scan of the chest or abdomen performed within 2 hours of admission. In 15 of these cases (42.8%), identification of the pneumothorax on CT scan resulted in alterations in management, including chest tube placement in 10 patients and intensified monitoring in 5 patients. Failure to identify pneumothoraces in trauma patients may lead to deterioration and significant complications in patients requiring anesthesia or mechanical ventilation. CT scan may facilitate identification in these cases.

Occlusion of left internal mammary artery with intra-aortic balloon: clinical implications.

Obstruction of the left internal mammary artery by a malpositioned intra-aortic balloon in patients undergoing coronary bypass grafting may erroneously preclude its use during the procedure or may cause a potentially fatal reduction in coronary flow postoperatively in patients with left internal mammary grafts.

Effect of phentolamine in controlling temperature and acidosis associated with cardiopulmonary bypass.

This study assessed the effects of phentolamine on rewarming patterns and metabolic acidosis in 37 patients subjected to hypothermia during cardiopulmonary bypass for the performance of aortocoronary bypass grafting. An additional 16 patients undergoing the same surgery received no phentolamine and served as a control group. In all patients, sodium bicarbonate (44.6 mEq) was administered only when the negative base excess was 3.0 mEq/L or greater. Sixty-eight percent of the patients receiving phentolamine and 56% of the control patients exhibited a uniform rewarming pattern in which the rectal, hand, and foot temperatures increased in parallel. In 32% of the patients receiving phentolamine and in 44% of the control patients, rectal and hand temperatures increased more than foot temperature during rewarming. Analysis of base excess values in the subgroups of patients with similar rewarming patterns indicated that base deficits were significantly decreased in patients receiving phentolamine. Phentolamine administration was also associated with significantly lower blood lactate levels and sodium bicarbonate requirements, as well as improvements in overall appearance and mental status. These data suggest that the routine use of phentolamine in patients undergoing cardiopulmonary bypass may be associated with more uniform body cooling and rewarming and improved tissue perfusion.

Influence of surgical technique on estrogen and progesterone receptor determinations in breast cancer.

The effect of surgical technique on hormone receptor values in breast cancer tissue was examined. One hundred and thirty-one women with breast cancer were divided into three groups: Group I, definitive diagnosis at frozen section and tissue taken for receptor determination; Groups II and III, diagnosis made by aspiration biopsy cytology, with tissue obtained from modified radical mastectomy in Group II and tissue obtained from mastectomy performed separately, prior to axillary lymph node dissection, in Group III. The rates of estrogen and progesterone receptor positivity, and the mean levels of receptor, did not differ significantly among the three groups. Thus tumor tissue from a mastectomy specimen can be reliably used for hormone receptor determinations, if the tumor tissue is kept cold and rapidly frozen for storage. Women whose cancer has been diagnosed by aspiration biopsy cytology need not undergo an additional open biopsy solely to obtain tissue for receptor determinations.

Use of Janke-Barron retractor for exposure of the arteries during bypass surgery.

The Janke-Barron heart support was used successfully on 1500 patients who required coronary artery bypass surgery to at least two vessels. The significant advantages it provided were improved exposure and access to all coronary arteries, elimination of damage caused by an assistant holding the heart, provision of a dryer operating field, and allowance for a smaller incision without compromising exposure of the heart.