Post-PKS Tailoring Steps of the Spiramycin Macrolactone Ring in Streptomyces ambofaciens

Spiramycins are clinically important 16-member macrolide antibiotics produced by Streptomyces ambofaciens. Biosynthetic studies have established that the earliest lactonic intermediate in spiramycin biosynthesis, the macrolactone platenolide I, is synthesized by a type I modular polyketide synthase (PKS). Platenolide I then undergoes a series of post-PKS tailoring reactions yielding the final products, spiramycins I, II, and III. We recently characterized the post-PKS glycosylation steps of spiramycin biosynthesis in S. ambofaciens. We showed that three glycosyltransferases, Srm5, Srm29, and Srm38, catalyze the successive attachment of the three carbohydrates mycaminose, forosamine, and mycarose, respectively, with the help of two auxiliary proteins, Srm6 and Srm28. However, the enzymes responsible for the other tailoring steps, namely, the C-19 methyl group oxidation, the C-9 keto group reduction, and the C-3 hydroxyl group acylation, as well as the timing of the post-PKS tailoring reactions, remained to be established. In this study, we show that Srm13, a cytochrome P450, catalyzes the oxidation of the C-19 methyl group into a formyl group and that Srm26 catalyzes the reduction of the C-9 keto group, and we propose a timeline for spiramycin-biosynthetic post-PKS tailoring reactions.     

Flux through the hexosamine pathway is a determinant of nuclear factor kappaB- dependent promoter activation

The hexosamine pathway may mediate some of the toxic effects of glucose. We hypothesized that flux through this pathway might regulate the activity of nuclear factor kappaB (NF-kappaB)-dependent genes in mesangial cells (MCs). In MCs, RT-PCR revealed that high glucose (30 mmol/l) and glucosamine (1 mmol/l) increased mRNA levels for vascular cell adhesion molecule 1 (VCAM-1) and increased the activity of an NF-kappaB enhancer by 1.5- and 2-fold, respectively. Overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for flux through the hexosamine pathway, led to a 2.2-fold increase in NF-kappaB enhancer activity; the combination of GFAT overexpression and high glucose increased activity 2.8-fold, and these increases were prevented by 40 micromol/l O-diazoacetyl-L-serine (azaserine) or 6-diazo-5-oxonorleucine. High glucose, glucosamine, and GFAT overexpression increased binding of MC nuclear proteins to NF-kappaB consensus sequences. Immunoblotting revealed that the p65 subunit of NF-kappaB was O-glycosylated in MC cultured in physiologic glucose and that significant enhancement occurred with high glucose and glucosamine. Both glucose and glucosamine dose-dependently increased human VCAM-1 promoter activity. In addition, GFAT overexpression activated the VCAM-1 promoter (2.25-fold), with further augmentation by high glucose and abrogation by inhibitors of GFAT, NF-kappaB, and O-glycosylation. Inactivation of the two NF-kappaB sites in the VCAM-1 promoter abolished its response to high glucose, glucosamine, and GFAT overexpression. These results suggest that increased flux through the hexosamine pathway leads to NF-kappaB-dependent promoter activation in MCs.     

Cytomegalovirus viremia during Campath-1H therapy for relapsed and refractory chronic lymphocytic leukemia and prolymphocytic leukemia

Campath-1H is effective therapy for patients with relapsed and refractory chronic lymphocytic leukemia (CLL) and prolymphocytic leukemia (PLL), but it is associated with profound lymphopenia and deficiencies in cell-mediated immunity. We report the incidence of cytomegalovirus (CMV) viremia in 34 patients treated with Campath-1H for relapsed or refractory CLL and PLL. All patients received infection prophylaxis during therapy and continuing for at least 2 months following Campath-1H. Five patients (15%) developed CMV viremia at a median of 28 days (range, 20-30 days) after the first dose of Campath-1H. The median CMV viral load was 860/mL (range, 420-2100/mL), as determined by quantitative plasma polymerase chain reaction (PCR). All 5 patients had a temperature > 38.5 degrees C, normal chest radiographs, normal liver function tests, and negative bacterial blood cultures with no clinical evidence of CMV disease at the time of presentation with CMV viremia. The median absolute neutrophil count (ANC) was 740/ microL (range, 340-1600/ microL), and the median absolute lymphocyte count (ALC) was 16/microL (range, 11-169/ microL) for the 5 patients at the time of CMV viremia. All 5 patients received ganciclovir therapy followed by prompt fever resolution and clearance of CMV viremia by plasma PCR. By univariate regression analysis, the following were not risk factors for CMV viremia: age, number of prior regimens, prior rituximab therapy, prior splenectomy, modified Rai stage at Campath-1H therapy (low/intermediate vs. high), ANC, and ALC; although, there was a trend towards significance for prior rituximab therapy (P = 0.07). Cytomegalovirus viremia may be a significant infectious complication during Campath-1H therapy and should be investigated further in future studies.     

Chronic telogen effluvium in a man

Chronic telogen effluvium is a recently described condition, in which there is persistent excessive hair shedding. Hairs are replaced as quickly as they shed, so patients never become bald. This condition is found primarily in women. We describe chronic telogen effluvium in a man; the diagnosis may have only become obvious because of his long hair.     

Breast cancer risk associated with different HRT formulations: a register-based case-control study

Background  

Previous epidemiological studies have inconsistently shown a modestly increased breast cancer risk associated with hormone replacement therapy (HRT). Limited information is available about different formulations - particularly concerning different progestins.     

Control of severe hemorrhage using C-clamp and arterial embolization in hemodynamically unstable patients with pelvic ring disruption

Introduction

Hemorrhage is the leading cause of death in patients with a pelvic fracture. The majority of blood loss derives from injured retroperitoneal veins and broad cancellous bone surfaces. The emergency management of multiply injured patients with pelvic ring disruption and severe hemorrhage remains controversial. Although it is well accepted that the displaced pelvic ring injury must be rapidly reduced and stabilized, the methods by which control of hemorrhagic shock is achieved remain under discussion. It has been proposed to exclusively use external pelvic ring stabilization for control of hemorrhage by producing a ‘tamponade effect’ of the pelvis. However, the frequency of clinically important arterial bleeding after external fixation of the pelvic ring remains unclear. We therefore undertook this retrospective review to attempt to answer this one important question: How frequently is arterial embolization necessary to control hemorrhage and restore hemodynamic stability after external pelvic ring fixation?

Materials and methods

We performed a retrospective review of 55 consecutive patients who presented with unstable types B and C pelvic ring fractures. Those patients designated as being in hemorrhagic shock (defined as a systolic blood pressure less than 90 mmHg after receiving 2 L of intravenous crystalloid) were treated by application of the pelvic C-clamp. Patients who remained in hemorrhagic shock, or were determined to be in severe shock (defined as mandatory catecholamines or more than 12 blood transfusions over 2 h), underwent therapeutic angiography within 24 h in order to control bleeding.

Results

Fourteen patients were identified as being hemodynamically unstable (ISS 30.1±11.3 points) and were treated with a C-clamp. In those patients with persistent hemodynamic instability, arterial embolization was performed. After C-clamp application, 5 of 14 patients required therapeutic angiography to control bleeding. Two patients died, one from multiple sources of bleeding and the other from an open pelvic fracture (total mortality 2/14, 14%).

Conclusions

Although the C-clamp is effective in controlling hemorrhage, one must be aware of the need for arterial embolization to restore hemodynamic stability in a select subgroup of patients.

     

Effect of ultraviolet light C on bacterial colonization in chronic wounds

Ultraviolet light C (light wavelength 200 nm to 290 nm) has been shown to kill cultures of antibiotic resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus. To evaluate the ability of ultraviolet light C to reduce the amount and type of bacteria present in chronically infected ulcers, as well as to establish the test-retest reliability of the semi-quantitative swab technique, a prospective, one-group, pre-post treatment study was conducted among patients receiving treatment in several in- and outpatient facilities and nursing homes. Individuals with chronic ulcers exhibiting at least two signs of infection and critically colonized with bacteria (n = 22) received a single 180-second treatment using an ultraviolet light C lamp (wavelength = 254 nm) placed 1 inch from the wound bed. Semi-quantitative swabs taken immediately before and after UVC treatment were used to assess changes in the bacterial bioburden present within the wound bed. Results demonstrated excellent test-retest reliability of the semi-quantitative swab technique used to evaluate the type and amount of bacteria present in chronic wounds (Cohen's kappa = 0.92). Assessment of wound bioburden using semi-quantitative swabs revealed a statistically significant (P <0.0001) reduction in the relative amount of bacteria following a single treatment of ultraviolet light C. The greatest reduction in semi-quantitative swab scores following ultraviolet light C treatment were observed for wounds colonized with the bacteria Pseudomonas aeruginosa and wounds colonized with only one species of bacteria. Significant (P <0.05) reductions in the relative amount of bacteria also were observed in 12 ulcers in which methicillin-resistant Staphylococcus aureus was present. These results confirm previous laboratory studies and demonstrate that ultraviolet light C can kill bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus present in superficial layers of chronic wounds.