Monitoring Cl- movement in single cells exposed to hypotonic solution.

Self-referencing ion--selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1-3 microm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (VN) measured in 100 and 10 mM NaCl (deltaVN = 57 +/- 2 mV), but was slightly greater than ideal when comparing 1 and 10 mM NaCl (deltaVN = 70 +/- 3 mV). The response was also linear in the presence of 1 mM glutamate, gluconate, or acetate, 10 microM tamoxifen, or 0.1, 1, or 10 mM HEPES at pH 7.0. The ISE was approximately 3 orders of magnitude more selective for Cl- over glutamate or gluconate but less than 2 orders of magnitude move selective for Clover bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was 'poisoned' by 50 microM 5-nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 +/- 5 mOsm) solution. The increase in efflux peaked 7-8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 microM tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level.

Volume-dependent glutamate permeation depends on transmembrane ionic strength and extracellular Cl-.

Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl(-) permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl(-) on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl(-) was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 m M) or low (50 m M) ionic strength solutions on both sides of the membrane, Cl(-) was always more permeable than Glu. When a transmembrane ionic strength gradient (150 m M extracellular: 50 m M intracellular) was set to drive water into the cell, and in the presence of extracellular Cl(-), Glu became up to 16-fold more permeable than Cl(-). Replacement of extracellular Cl(-) with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity.

Surgical smoke: a review of the literature. Is this just a lot of hot air?

Surgical smoke is omnipresent in the day-to-day life of the surgeon and other medical personnel who work in the operating room. In addition, patients are also exposed, especially and uniquely so in laparoscopic cases where smoke is created and trapped in a closed and absorptive space. Surgical smoke has typically been produced by electrocautery but is now ever more present in a new form with the burgeoning use of the laser and the harmonic scalpel. Several cases of transmission of human papillomavirus (HPV) from patient to treating professional via laser smoke have alerted us to the reality that surgical smoke in certain situations is far form benign. However, surgeons rarely take measures to protect themselves, their co-coworkers and patients from surgical smoke. Should we and, if so, how do we differentiate between different types of smoke and should we move toward increasing our efforts to protect ourselves, our co-workers, and patients from it? This article attempts to sort through the available data and draw some reasonable conclusions regarding surgical smoke. In general, surgical smoke is a biohazard and cannot be ignored. At a minimum, surgical smoke is a toxin similar to cigarette smoke. However, other dangers exist. This is especially true in specific circumstances such as when tissue infected with dangerous viruses is aerosolized by lasers. In addition, smoke generated by the harmonic scalpel, being a relatively cold vapor similar to laser smoke, should be further investigated for its potential ill effects and until then, looked upon with reasonable caution. Although not a high-priority in most surgical cases, surgeons should support efforts to minimize OR personnel, patients, and their own exposure to surgical smoke.

DPC 681 and DPC 684: potent, selective inhibitors of human immunodeficiency virus protease active against clinically relevant mutant variants.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) are important components of many highly active antiretroviral therapy regimens. However, development of phenotypic and/or genotypic resistance can occur, including cross-resistance to other PIs. Development of resistance takes place because trough levels of free drug are inadequate to suppress preexisting resistant mutant variants and/or to inhibit de novo-generated resistant mutant variants. There is thus a need for new PIs, which are more potent against mutant variants of HIV and show higher levels of free drug at the trough. We have optimized a series of substituted sulfonamides and evaluated the inhibitors against laboratory strains and clinical isolates of HIV type 1 (HIV-1), including viruses with mutations in the protease gene. In addition, serum protein binding was determined to estimate total drug requirements for 90% suppression of virus replication (plasma IC(90)). Two compounds resulting from our studies, designated DPC 681 and DPC 684, are potent and selective inhibitors of HIV protease with IC(90)s for wild-type HIV-1 of 4 to 40 nM. DPC 681 and DPC 684 showed no loss in potency toward recombinant mutant HIVs with the D30N mutation and a fivefold or smaller loss in potency toward mutant variants with three to five amino acid substitutions. A panel of chimeric viruses constructed from clinical samples from patients who failed PI-containing regimens and containing 5 to 11 mutations, including positions 10, 32, 46, 47, 50, 54, 63, 71, 82, 84, and 90 had mean IC(50) values of <20 nM for DPC 681 and DPC 681, respectively. In contrast, marketed PIs had mean IC(50) values ranging from 200 nM (amprenavir) to >900 nM (nelfinavir).

Relaxin decreases renal interstitial fibrosis and slows progression of renal disease.

BACKGROUND: Relaxin, a hormone of the insulin-growth factor family, promotes collagen remodeling. In rodent models of pulmonary and dermal fibrosis, relaxin reduced interstitial fibrosis. To study relaxin's effect in renal disease, we used the experimental bromoethylamine (BEA) model that leads to severe renal interstitial fibrosis, a decrease in glomerular filtration rate, and albuminuria at one month. METHODS: Rats were injected with BEA one week prior to implantation of an osmotic pump delivering relaxin (2 microg/hour) or vehicle continuously for 28 days. RESULTS: BEA caused a significant decrease in creatinine clearance, which was partially prevented by relaxin. In the relaxin-treated BEA rats, serum creatinine was normal, and albumin excretion was slightly decreased. By morphometric measurement, relaxin administration was associated with a significant decrease in interstitial fibrosis at the corticomedullary junction. This was accompanied by a decrease in the number of ED-1 positive cells (an index of macrophage infiltration) and in the intensity of immunohistochemical staining for transforming growth factor-beta. This antifibrotic effect of relaxin did not appear to be mediated by systemic hemodynamic changes since the mean arterial pressure was not significantly different among the groups. CONCLUSIONS: Relaxin may have a useful application in decreasing interstitial fibrosis and thereby slowing the progression of renal disease.

The Fc receptor for IgG expressed in the villus endothelium of human placenta is Fc gamma RIIb2.

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fc gamma receptor (Fc gamma R) expression by immunohistology and immunoblotting. Several pan-Fc gamma RII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical Fc gamma RIIa were not expressed in transport-related endothelium of the placenta. Our inference, that Fc gamma RIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of Fc gamma RIIb. These experiments showed that the Fc gamma RII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.

3,3a-Dihydropyrano[4,3,2-de]quinazolin-2(1H)-ones are potent non-nucleoside reverse transcriptase inhibitors.

A series of unique 3,3a-dihydropyrano[4,3,2-de]quinazolin-2(1H)-ones and a 2a,5-dihydro-2H-thieno[4,3,2-de]quinazo-line-4(3H)-thione were found to be HIV-1 non-nucleoside reverse transcriptase inhibitors. One of these compounds, as the racemate, possessed an IC90 = 4.6 nM against wild-type virus in a whole cell antiviral assay and had an IC90 = 76 and 897 nM against the clinically significant K103N and K103N/L100I mutant viruses, respectively.

Differential effects of enalapril and irbesartan in experimental papillary necrosis.

This study was undertaken to determine whether angiotensin receptor blockers are as renoprotective as angiotensin-converting enzyme inhibitors in an experimental model of chronic interstitial renal disease. Groups of rats received one of the following treatments for 1 week: (1) enalapril, (2) diltiazem, (3) a cocktail of hydralazine, reserpine, and hydrochlorothiazide, or (4) irbesartan (an AT1 antagonist). The animals were injected with bromoethylamine (200 mg/kg), and antihypertensive treatment continued for 1 month. All drugs were effective in lowering the mean arterial pressure. The bromoethylamine-treated rats developed albuminuria and sustained a 40-50% decrease in creatinine clearance. Enalapril and irbesartan reduced albuminuria, but only enalapril partially prevented the decline in creatinine clearance and lowered the number of ED-1-positive cells. Diltiazem and cocktail had no effect on proteinuria, creatinine clearance, or ED-1 cells. In this experimental model, the effects of enalapril and irbesartan were not identical. Both drugs reduced proteinuria, but enalapril was more effective in protecting the renal function. The fact that the AT1 antagonist protected against albuminuria but did not affect the clearance of creatinine implies that the results seen with angiotensin-converting enzyme inhibition may be in part due to an effect on angiotensin II via AT2 receptor blockade or through an effect on bradykinin.

Drug design with a new transition state analog of the hydrated carbonyl: silicon-based inhibitors of the HIV protease.

BACKGROUND: Silicon is the element most similar to carbon, and bioactive organosilanes have therefore been of longstanding interest. Design of bioactive organosilanes has often involved a systematic replacement of a bioactive molecule's stable carbon atoms with silicon. Silanediols, which are best known as unstable precursors of the robust and ubiquitous silicone polymers, have the potential to mimic an unstable carbon, the hydrated carbonyl. As a bioisostere of the tetrahedral intermediate of amide hydrolysis, a silanediol could act as a transition state analog inhibitor of protease enzymes. RESULTS: Silanediol analogs of a carbinol-based inhibitor of the HIV protease were prepared as single enantiomers, with up to six stereogenic centers. As inhibitors of this aspartic protease, the silanediols were nearly equivalent to both their carbinol analogs and indinavir, a current treatment for AIDS, with low nanomolar K(i) values. IC(90) data from a cell culture assay mirrored the K(i) data, demonstrating that the silanediols can also cross cell membranes and deliver their antiviral effects. CONCLUSIONS: In their first evaluation as inhibitors of an aspartic protease, silanediol peptidomimetics have been found to be nearly as potent as currently available pharmaceutical agents, in enzyme and cell protection assays. These neutral, cell-permeable transition state analogs therefore provide a novel foundation for the design of therapeutic agents.

Infant mortality and early postpartum discharge.

OBJECTIVE: To assess additional risk of newborn death owing to early discharge. METHODS: This was a historical cohort study using Washington State linked birth certificates, death certificates, and hospital discharge records that covered 47,879 live births in 1989 and 1990. Logistic regression was used to assess risk of death within the first year of life after early discharge (less than 30 hours after birth) compared with later discharge (30-78 hours after birth). RESULTS: Newborns discharged early were more likely to die within 28 days of birth (odds ratio [OR] 3.65; 95% confidence interval [CI] 1.56, 8.54), between 29 days and 1 year (OR 1.61; 95% CI 1.10, 2.36), and any time within the first year (OR 1.84; 95% CI, 1.31, 2.60) of life than newborns sent home later. Newborns discharged early also were more likely to die of heart-related problems (OR 3.72; CI 1.25, 11.04) and infections (OR 4.72; CI 1.13, 19.67) within 1 year of birth than newborns discharged later. CONCLUSION: Newborns discharged within 30 hours of birth are at increased risk of death within the first year of life.

Sexuality in persons with lower extremity amputations.

PURPOSE: There is a paucity of information regarding sexual functioning in persons with lower extremity amputations. The purpose of this study was to describe sexual and psychological functioning and health status in persons with lower extremity amputation. METHODS: Self-report surveys assessed sexual functioning (Derogatis Inventory), depression (Beck Depression Inventory, anxiety (State-Trait Anxiety Inventory), and health status (Health Status Questionnaire) in a convenience sample of 30 men with lower extremity amputations. Mean age of the participants was 57 years (range 32-79). Mean duration since amputation was 23 months (range 3-634 months). Twenty one subjects (70%) had trans-tibial and seven subjects (23%) had trans-femoral amputations. RESULTS: A majority of subjects were experiencing problems in several domains of sexual functioning. Fifty three percent (n = 16) of the subjects were engaged in sexual intercourse or oral sex at least once a month. Twenty seven percent (n = 8) were masturbating at least once a month. Nineteen subjects (63%) reported orgasmic problems and 67% were experiencing erectile difficulties. Despite these problems, interest in sex was high in over 90% of the subjects. There was no evidence of increased prevalence of depression or anxiety in these subjects when compared to other outpatient adult populations. CONCLUSIONS: Sexual problems were common in the subjects studied. Despite these problems, interest in sex remained high. Few investigations have been directed toward identifying the psychological and social factors that may contribute to these problems and more research with a larger population is needed in this area.

Modulation of volume regulated anion current by I(Cln).

I(Cln), a cytosolic protein associated with a nucleotide-sensitive chloride current, may be involved in the regulation of a volume-regulated anion current (VRAC) associated with hypotonic cell swelling. We have determined the nucleic acid sequences of I(Cln) from human tsA201a, colonic (T84) and myeloma (RPMI 8826) cell lines. The amino acid sequences are highly homologous (>/=99%) to each other but less homologous to I(Cln) protein from other species. Using the whole-cell patch clamp technique, we examined the effect of I(Cln) protein expression levels on VRAC properties during a hyposmotic challenge. Overexpression of T84 or RPMI 8226-derived I(Cln) protein in tsA201a cells results in a more than 9-fold increase in the rate of VRAC activation over control values, while having no effect on VRAC inactivation properties. Underexpression of endogenous I(Cln) protein in tsA201a cells using antisense oligonucleotides results in a more than 180-fold decrease in VRAC activation rate as compared to control values. These results indicate that I(Cln) protein expression modulates VRAC activation but not inactivation.

Inhibition of clinically relevant mutant variants of HIV-1 by quinazolinone non-nucleoside reverse transcriptase inhibitors.

A series of 4-alkenyl and 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones were found to be potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type-1 (HIV-1). The 4-alkenyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 082 and DPC 083 and the 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 961 and DPC 963 were found to exhibit low nanomolar potency toward wild-type RF virus (IC(90) = 2.0, 2.1, 2.0, and 1.3 nM, respectively) and various single and many multiple amino acid substituted HIV-1 mutant viruses. The increased potency is combined with favorable plasma serum protein binding as demonstrated by improvements in the percent free drug in human plasma when compared to efavirenz: 3.0%, 2.0%, 1.5%, 2. 8%, and 0.2-0.5% for DPC 082, DPC 083, DPC 961, DPC 963, and efavirenz, respectively.

Pressure ulcer risk in spinal cord injury: predictors of ulcer status over 3 years.

OBJECTIVE: To identify predictors of pressure ulcers in men with spinal cord injury over a 3-year period. DESIGN: Longitudinal, two-panel, cohort. SETTING: Community. PARTICIPANTS: One hundred eighteen men with spinal cord injury. MEASURES: Interviews, questionnaires, and physical examinations were completed in two phases, 3 years apart. Information obtained included demographic and spinal cord injury characteristics; ulcer history; health beliefs and practices; measures of impairment, disability, and handicap; and skin integrity. RESULTS: Thirty-one percent of the participants reported having a pressure ulcer in the 12 months before Phase 2. Some Phase 1 predictors of self-reported ulcers in the year before Phase 2 were a younger age at onset of spinal cord injury, previous pressure ulcer surgery, and the presence of a pressure ulcer in the year before Phase 1. On examination at Phase 2, 59% presented with an ulcer. Phase 1 predictors of ulcer presence at Phase 2 examination were similar to predictors for self-reported ulcers. CONCLUSION: Individuals with the identified predictive characteristics are at greater risk for developing pressure ulcers. These individuals should receive additional interventions to reduce that risk. Potential interventions include more systematic and frequent follow-up, frequent review of pressure ulcer prevention and management strategies, and provision of needed personal assistance and relevant equipment.

Jejunal diverticulosis: a rare entity with multiple presentations, a series of cases.

BACKGROUND/AIMS: Jejunal diverticulosis (JD) is a rare disease that has a variable presentation. The signs, symptoms, diagnosis, complications and treatment of JD will be discussed through a review of the literature and a series of cases from our own institution. METHODS: A retrospective analysis of the diagnosis, treatment and complications of JD was performed by a literature review. This was accompanied by a series of four cases of JD diagnosed and treated in our own institution. CONCLUSIONS: JD is a rare disease in which most patients are asymptomatic. However, JD's different complications are serious and can be fatal. The treatment is mainly surgical; however, there have been cases where nonsurgical management was successful.

Decreased cerebrospinal fluid absorption during abdominal insufflation.

BACKGROUND: Intracranial pressure (ICP) is known to rise during induced CO(2) pneumoperitoneum. This rise correlates with an increase in inferior vena caval pressure; therefore, it is probably associated with increased pressure in the lumbar venous plexus. Branches of this plexus communicate with arachnoid villi in the lumbar cistern and the dural sleeves of spinal nerve roots-areas where cerebrospinal fluid (CSF) absorption to normally takes place. The increased venous pressure in this area may impede CSF absorption. Because CSF is produced at a constant rate, decreased absorption will increase ICP. We hypothesized that increased ICP occurring during abdominal insufflation is due, at least in part, to decreased absorption of CSF. The purpose of this study is to show that CSF absorption is inhibited during abdominal insufflation. METHODS: After appropriate approval was obtained, 16 domestic swine were anesthetized and injected into the CSF with 100 microcuries (microCu) of I(131) radioactive iodinated human serum albumin (RISA) in 2 ml of normal saline. Eight subjects underwent CO(2) abdominal insufflation to 15 mmHg and were maintained for 4 h. A control group did not undergo insufflation. Blood levels of RISA were measured over a 4-h period to determine the rate of CSF absorption. RESULTS: Blood levels of RISA increased at a slower rate in the subjects undergoing abdominal insufflation than in the control group. The mean change over 2 h in the insufflated group was 15% compared to 34% in the control group (p = 0.02). This difference indicates decreased absorption of CSF in the insufflated group. CONCLUSIONS: These results demonstrate decreased absorption of CSF during abdominal insufflation and support the hypothesis that the increase in ICP pressure occurring during abdominal insufflation is caused, at least in part, by decreased absorption of CSF in the region of the lumbar cistern and the dural sleeves of spinal nerve roots.

Changes in collagenases and TGF-beta precede structural alterations in a model of chronic renal fibrosis.

BACKGROUND: To study the role of collagenases and transforming growth factor-beta (TGF-beta) in the genesis of interstitial fibrosis, we used the model of bromoethylamine (BEA)-induced papillary necrosis, which is known to lead over a period of 1 to 12 months to interstitial fibrosis and renal insufficiency. METHODS: Rats were injected with BEA, and urine and kidney tissue (cortex and medulla) were collected after 1, 2, 3, 7, and 30 days. One kidney was perfused and fixed for morphological studies and immunostained for collagen type I, III, and IV. The other kidney was used to prepare cortex and medulla extracts for gelatinases (by fluorometric and zymographic techniques), tissue inhibitors of metalloproteinase-1 (TIMP-1), and TIMP-2 (by enzyme-linked immunosorbent assay, ELISA) and TGF-beta1 (by ELISA). RESULTS: Albuminuria and interstitial fibrosis were present in BEA rats by day 7, which continued until day 30. Immunocytochemical staining for collagen types showed that collagen III and IV increased in the interstitium by day 30, but collagen I remained unchanged. Gelatinase activity in the medulla decreased by 57% compared with control by day 2 and remained low until day 30. In the cortex, gelatinase activity remained unchanged between 0 and 7 days after BEA but decreased by 72% by day 30. TIMP-1 and TIMP-2 were decreased by 80% compared with day 0 in both the medulla (by day 1) and cortex (by day 2) and remained low up to day 30. TGF-beta1 immunoreactivity increased progressively until day 2 in the medulla (16-fold higher than control) and day 3 in the cortex (8-fold higher than control) and returned to control level by day 3 in the medulla and by day 30 in the cortex. Two days after BEA injection, the mRNA for TGF-beta1 was increased eightfold in the cortex and 12-fold in the medulla, and it remained high for up to 30 days. CONCLUSIONS: The fibrosis that follows papillary necrosis is associated with both high TGF-beta1 expression and depressed gelatinolytic activity.