The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells.

Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5'GCGCGC3') or an arbitrary (5'ACGCGT3') palindrome sequence in place of the 39-nucleotide DIS stem-loop (NL(CGCGCG) and NL(ACGCGT)). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1.

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

An analysis of breast cancer surgery after free transverse rectus abdominis myocutaneous (TRAM) flap reconstruction.

BACKGROUND: Breast reconstruction is currently offered on a more routine basis to patients after mastectomy for breast cancer. This paper analyzes the outcomes of breast cancer surgery, and the results and effects of breast reconstruction using free TRAM flaps. METHODS: A retrospective review of 75 consecutive patients who had free transverse rectus abdominis myocutaneous (TRAM) flap breast reconstruction after breast cancer surgery was performed. A total of 92 free TRAM flaps were performed on 75 patients in Victoria, British Columbia, from January 1992 to May 1999. Thirty-three patients (44%) underwent primary breast cancer surgery and an immediate reconstruction (7 bilateral and 27 unilateral) and 42 patients (56%) had delayed reconstruction (10 bilateral and 32 unilateral). RESULTS: Twenty- one patients (28%) had stage 0 disease, 20 (26.7%) had stage I disease, 17 (22.7%) had stage IIA disease, 12 (15%) had stage IIB disease, and 4 (5.3%) had stage IIIA disease. In 1 patient the stage of disease was unknown. The mean patient age was 49.4 years (range 33 to 73). Of the patients undergoing immediate reconstruction 3 had postoperative chemotherapy and 1 had postoperative radiotherapy. Three patients had combined chemoradiotherapy. In none of these cases was the adjuvant therapy delayed by the reconstructive surgery. Overall mean follow-up time from cancer diagnosis was 56.8 months and from the time of TRAM flap reconstruction, 36.7 months. To date, 5 recurrences have been detected (6.6%). Mean time between reconstruction and detection of recurrence was 22.8 months. Detection of recurrence was achieved clinically and was not impaired in any of the cases by the presence of the free flap. Patient satisfaction was assessed via a telephone survey, with 93% of patients pleased with the cosmetic results of their surgery. CONCLUSIONS: For those patients with breast cancer requiring mastectomy, free TRAM flap reconstruction is a safe, cosmetically acceptable surgical alternative that impairs neither effective breast cancer surgery nor detection of recurrent disease.

Effects of treadmill exercise on sweating in three breeds of goats.

OBJECTIVE: To compare the sweating responses of three breeds of goats to exercise at 30 degrees C. DESIGN: Factorial experiment with two goats of each of three breeds exercised for 60 min at 3 km/h and 30 degrees C on 6 days. PROCEDURE: Two mature females of the Anglo-Nubian, Saanen and Toggenburg breeds were used. Rectal temperature, respiration rate and sweating rate at three sites were recorded every 20 min during six replicates of exercise at 48 h intervals on a treadmill. RESULTS: Respiration rate varied with time and breed (P < 0.01) and increased from 20 +/- 2 breaths/min to 135, 195 and 260/min after 60 min exercise in Anglo-Nubian, Saanen and Tottenburg goats, respectively. Bread differences in rectal temperature were small but significant (P < 0.001), and mean values increased from 38.9 degrees C before exercise to 39.7 degrees C after 1 h exercise. The breed x sites interaction for sweating was significant (P < 0.01). On the rump breed differences in sweating rate were not significant. On the loin, Toggenburg goats started to sweat most rapidly and reached maximal values of 80.2 +/- 10.6 g/m2/h after 20 min and then decreased to 70.0 +/- 4.3 g/m2/h at 60 min. Sweating rate on the ear was highest in Toggenburg goats, followed by those of Saanen and Anglo-Nubian goats (P < 0.05), but the differences were small (7 g/m2/h); peak values of 67.3 to 76.1 g/m2/h were recorded after 20 min. CONCLUSION: Respiration and sweating rates increased significantly during exercise in all three breeds of goats, but breed differences were marked only for respiration rate. The goats sweated more on the rump than on the loin on ear, with peak values after 40 min of exercise.

Identification of disease response genes expressed in Gossypium hirsutum upon infection with the wilt pathogen Verticillium dahliae.

Verticillium wilt is a vascular disease of cotton (Gossypium spp.) caused by the fungal pathogen Verticillium dahliae. To begin to understand the molecular mechanisms of the disease response in cotton cultivars that display superior wilt tolerance, such as Gossypium hirsutum cv. Sicala V-1, a cDNA library was constructed with mRNA isolated from root tissue of Sicala V-1, 24 h after inoculation with V. dahliae. The library was screened by a differential screening technique which was successful in identifying differences in gene expression between uninfected and V. dahliae-infected G. hirsutum root tissue. Among the differentially expressed clones, 51% represented up-regulated genes which had the potential to be involved in the defence response of G. hirsutum. The temporal expression patterns of nine suspected defence response genes were examined by northern blot analysis at several time intervals after inoculation with V. dahliae. The rapid increase in mRNA transcripts corresponding to each of these clones upon infection suggests a role for these genes in the defence response of G. hirsutum. Genes not previously associated with the defence response of the cotton plant, such as those for a 14-3-3-like protein and pathogenesis-related (PR) proteins, have been identified together with presumably novel genes, for which a definite function could not be ascribed.

Skin and soft tissue infections in critical care.

Clinicians in the critical care setting see extensive, severe, and often necrotizing infection, as well as atypical or unusual infection that can be misdiagnosed, resulting in delays in treatment. Greater numbers of immunocompromised patients are at risk for both types of infection. A thorough understanding of various etiologies and presentations of these infections is essential, as is the ability to discriminate between infectious and noninfectious etiologies.

Leptospiral pneumonia.

Leptospirosis, a spirochetal infection, causes a wide spectrum of disease ranging from asymptomatic infection, or influenza-like symptoms, to severe jaundice and renal failure. Humans become infected through skin or mucous membrane contact with infected animal urine or urine-contaminated water or soil. The most common source of human infection worldwide is rats. However, in the United States, dogs, livestock, wild mammals, and cats are also sources. Once leptospires penetrate mucous membranes or breaks in the skin, they disseminate to all parts of the body. Five to ten percent of those infected will have severe leptospirosis with jaundice, known as Weil's disease. The classical presentation of leptospirosis is that of a biphasic illness. The initial septicemic phase lasts 4 to 7 days and is characterized most commonly as a mild influenza-like illness. During the secondary immune phase, leptospires disappear from the blood and cerebrospinal fluid. However, circulating antibodies cause immune-mediated meningitis, uveitis, rash, and, very rarely, circulatory collapse associated with Weil's disease. Pulmonary involvement occurs in 20% to 70% of patients. The more severe pulmonary manifestations are rare. Although attempts should be made to isolate leptospires from the blood or cerebrospinal fluid, the diagnosis is usually established by serologic tests. The effectiveness of antimicrobial therapy in treating leptospirosis has been difficult to assess because of the high variability of the disease's clinical course, although in severe cases, antibiotic therapy is effective even when treatment is delayed. Prevention is difficult because it is almost impossible to eliminate the large animal reservoir of infection.

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare.

Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M intracellulare that is absent in M. avium. This antigen cross reacts immunologically with a major 38 kDa antigen of M. tuberculosis, and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli. Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.

Aeromonas hydrophila infections following use of medicinal leeches in replantation and flap surgery.

Aeromonas hydrophila infections are a recognized complication of postoperative leech application, and can occur with measurable frequency in populations of patients treated with leeches. We review 11 previously reported leech-related Aeromonas infections and analyze seven unreported cases. These infections range from minor wound complications to extensive tissue loss and sepsis. Often, these infections followed leech application to tissue with questionable arterial perfusion. Onset of clinical infection in these patients ranged from within 24 hours of leech application to 10 days or more after leech application. Late infections may represent bacterial invasion from colonized necrotic tissue. Based on these observations, we recommend that leech applications be restricted to tissue with arterial perfusion to minimize contamination of necrotic tissue. We also recommend that patients treated with leeches receive antibiotics effective against Aeromonas hydrophila before leech application. Patients treated with leeches and discharged with eschars or open wounds might benefit from oral antibiotic therapy until wound closure. These precautions may minimize or eliminate this complication of leech use.

Anaerobic disease of the lung.

Anaerobic pleuropulmonary infections present in various ways. Aspiration pneumonitis occurs first and may be quite difficult to distinguish from the acute bacterial pneumonia caused by Streptococcus pneumoniae and other organisms. Although aspiration pneumonitis may be self-limiting, sequelae such as lung abscess, necrotizing pneumonia, and empyema can develop. Empiric antimicrobial therapy is now acceptable. The initial choice of antimicrobial agents is based on which pathogens are likely to be involved.

Skin surface electron microscopy in Pityrosporum folliculitis. The role of follicular occlusion in disease and the response to oral ketoconazole.

The yeast Pityrosporum orbiculare is thought to cause the folliculitis associated with seborrheic eczema. However, a combination of mechanical and microbiological factors may be involved, with follicular occlusion leading to yeast overgrowth and folliculitis. Scanning electron microscopy was used to investigate this hypothesis. Skin biopsy specimens obtained from patients with Pityrosporum folliculitis were examined by scanning electron microscopy before and after oral ketoconazole therapy. Patients with active disease showed occlusion of noninflamed follicles, which resolved after ketoconazole treatment. Follicular occlusion was not present in biopsy specimens obtained from unaffected controls nor was it related to the presence of P orbiculare. These findings suggest that follicular occlusion may be a primary event in the development of this folliculitis, with yeast overgrowth a secondary occurrence. The beneficial effect of ketoconazole in this disease may be due to direct effects on the follicle.

Skin surface electron microscopy in Pityrosporum folliculitis. The role of follicular occlusion in disease and the response to oral ketoconazole.

The yeast Pityrosporum orbiculare is thought to cause the folliculitis associated with seborrheic eczema. However, a combination of mechanical and microbiological factors may be involved, with follicular occlusion leading to yeast overgrowth and folliculitis. Scanning electron microscopy was used to investigate this hypothesis. Skin biopsy specimens obtained from patients with Pityrosporum folliculitis were examined by scanning electron microscopy before and after oral ketoconazole therapy. Patients with active disease showed occlusion of noninflamed follicles, which resolved after ketoconazole treatment. Follicular occlusion was not present in biopsy specimens obtained from unaffected controls nor was it related to the presence of P orbiculare. These findings suggest that follicular occlusion may be a primary event in the development of this folliculitis, with yeast overgrowth a secondary occurrence. The beneficial effect of ketoconazole in this disease may be due to direct effects on the follicle.

Pulmonary sporotrichosis with massive hemoptysis.

Pulmonary sporotrichosis is an uncommon condition that may mimic tuberculosis. We present a patient who, years after antifungal therapy, presented with massive pulmonary hemorrhage. Although such life-threatening hemoptysis caused by sporotrichosis is rare, the possibility of this complication of progressive disease should influence decisions regarding medical and surgical treatment of patients with extensive cavitary involvement.

Brain abscess from chronic odontogenic cause: report of case.

A case of a chronic odontogenic abscess that probably precipitated a frontopatrietal brain abscess by hematogenous spread has been presented. A thorough examination of the patient showed no other source of infection. Peptostreptococcus sp was found as the common pathogen despite 6 days of broad-spectrum antibiotic therapy. This case emphasizes the important role of dentistry in medical diagnosis and treatment implemented in the hospital setting.

Principles of antimicrobial therapy for head and neck infections.

The physician should take a logical, systematic approach when choosing antimicrobial therapy. The recent proliferation of antimicrobial agents and changing susceptibility patterns amongst bacterial pathogens have made this a more arduous task. Classifying antibiotics according to their spectrum of activity, tissue penetration and potential side effects will aid in choosing empiric therapy for serious head and neck infections.

Localized and systemic infection due to Vibrio species.

Many important species have been added to the genus Vibrio in the past several years. Vibrios have been associated with a wide variety of clinical syndromes ranging from mild gastroenteritis to life-threatening cellulitis. Most Vibrio infections follow consumption of raw shellfish or exposure to sea water. Although much has been learned about these organisms in the past several years, additional information is needed concerning pathogenesis, diagnosis, treatment, and prevention of Vibrio infections.

ECG abnormalities in myoglobinuria: review of the literature.

Reports of electrocardiographic abnormalities in association with myoglobinuria have been sparse and have included conduction disturbances, ST segment shifts, and T-wave changes. In many instances, these changes were noted in patients with underlying heart disease. We report a case of a 34-year-old woman with ST segment depression and T-wave inversion in the inferolateral leads during the acute episode of myoglobinuria. There was no demonstrable underlying heart disease, and there was parallel resolution of these ECG changes with myoglobinuria. We conclude that these ECG changes were produced by cardiac muscle involvement in a manner similar to that observed in skeletal muscle in myoglobinuria.

The effects of defaunation of the rumen on the growth of lambs on low-protein-high-energy diets.

1. The effects of defaunation of the rumen of lambs given low-protein-high-energy diets were studied using animals fed ad lib. a basal diet of sugar and oaten chaff which was supplemented with fish meal at various levels. These diets supported relatively large populations of protozoa in the rumen. 2. A nonyl phenol ethoxylate (15 g. Teric GN9:ICI Australian Ltd) given intraruminally, was shown to be an effective means of defaunating the rumen and the lambs were maintained protozoa-free by isolation. 3. In lambs given low levels of fish meal in the diet, the absence of rumen protozoa increased growth rate and improved the food conversion efficiency (g dry matter intake/g growth). These effects were not apparent at high levels of protein supplementation. 4. These results suggested that the absence of rumen protozoa resulted in an increased availability of nutrients supplying both energy and amino acids since the efficiency of food utilization and the rate of wool growth (which is sensitive to amino acid supply) were increased in lambs on the low level of protein supplementation without apparently increasing food intake.

Dibutyryl cyclic AMP, adenosine and guanosine blockade of the dopamine, ergocryptine and apomorphine inhibition of prolactin release in vitro.

Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.