Identification of Candida species in the clinical laboratory: a review of conventional, commercial, and molecular techniques.

In healthy individuals, Candida species are considered commensal yeasts of the oral cavity. However, these microorganisms can also act as opportunist pathogens, particularly the so-called non-albicans Candida species that are increasingly recognized as important agents of human infection. Several surveys have documented increased rates of C. glabrata, C. tropicalis, C. guilliermondii, C. dubliniensis, C. parapsilosis, and C. krusei in local and systemic fungal infections. Some of these species are resistant to antifungal agents. Consequently, rapid and correct identification of species can play an important role in the management of candidiasis. Conventional methods for identification of Candida species are based on morphological and physiological attributes. However, accurate identification of all isolates from clinical samples is often complex and time-consuming. Hence, several manual and automated rapid commercial systems for identifying these organisms have been developed, some of which may have significant sensitivity issues. To overcome these limitations, newer molecular typing techniques have been developed that allow accurate and rapid identification of Candida species. This study reviewed the current state of identification methods for yeasts, particularly Candida species.

Molecular fingerprinting methods for the discrimination between C. albicans and C. dubliniensis.

Opportunistic fungal pathogens are becoming increasingly important causes of both community-acquired and nosocomial infections. The most important fungal pathogens are yeast species belonging to the genus Candida. These species show differences in levels of resistance to antifungal agents and mortality. Consequently, it is important to correctly identify the causative organism to the species level. Identification of Candida dubliniensis in particular remains problematic because of the high degree of phenotypic similarity between this species and Candida albicans. However, as the differences between both are most pronounced at the genetic level, several studies have been conducted in order to provide a specific and rapid identification fingerprinting molecular test. In most candidal infectious, no single DNA fingerprinting technique has evolved as a dominant method, and each method has its advantages, disadvantages and limitations. Moreover, the current challenge of these techniques is to compile standardized patterns in a database for interlaboratory use and future reference. This review provides an overview of most common molecular fingerprinting techniques currently available for discrimination of C. albicans and C. dubliniensis.

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.

In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa.

OBJECTIVE: To compare the outcomes of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) with fresh and cryopreserved testicular spermatozoa in patients with obstructive and nonobstructive azoospermia. DESIGN: Retrospective analysis of consecutive ICSI cycles. SETTING: Large urban reproductive medicine program. PATIENT(S): Twenty-nine patients with obstructive and nonobstructive azoospermia undergoing testicular sperm extraction for a total of 46 IVF-ICSI cycles (12 fresh, 34 frozen). INTERVENTION(S): Testicular sperm extraction, cryopreservation, and IVF-ICSI with fresh or frozen-thawed spermatozoa. MAIN OUTCOME MEASURE(S): Fertilization rates, embryo cleavage rates, embryo implantation rates, clinical pregnancy rates per cycle and per embryo transfer, and delivery and spontaneous abortion rates. RESULT(S): No statistically significant differences were noted in any of the parameters examined between IVF-ICSI cycles from fresh or frozen-thawed testicular spermatozoa. Fertilization rates were 56% with fresh vs. 61% with frozen-thawed testicular sperm, cleavage rates 92% vs. 95%, implantation rates 26% vs. 17%, clinical pregnancy rates per cycle 33% vs. 41%, and pregnancy rates per embryo transfer 33% vs. 45%, respectively. Delivery rates were 75% with fresh vs. 69.2% with frozen-thawed testicular sperm, and spontaneous abortion rates 25% and 30.8%, respectively. CONCLUSION(S): No differences were found in IVF-ICSI outcomes between cryopreserved and fresh testicular sperm. In addition, cryopreservation provides several advantages for the patients and reproductive team.

A pitch glide activates an intermediate response between auditory N1 and mismatch negativity.

Pitch glides of a continuous tone elicit auditory N1-like responses. However, their characteristics have not well been investigated, and it remained unclear whether the response is an auditory true N1 or the mismatch negativity (MMN). We found here that a rapid pitch glide activates almost the same response as a true N1. On the contrary, as the rate of the pitch glide decreases, the response continuously varies the characteristics from true N1 to MMN. This suggests that there would exist intermediate responses between auditory N1 and MMN.

Hoxa-10 deficient male mice exhibit abnormal development of the accessory sex organs.

The role of mammalian Hox genes in regulating segmental patterning of axial structures and the limb is well established. A similar role in development of soft tissue organ systems has recently been suggested by observations linking several 5' members of the HoxA and HoxD clusters to segmentation events and morphogenesis in the gastrointestinal and genitourinary systems. We have specifically examined the role of Hoxa-10 in development of the male accessory sex organs by characterizing expression of Hoxa-10 in the developing male reproductive tract and correlating expression to morphologic abnormalities in knockout mice deficient for Hoxa-10 function. We report that Hoxa-10 expression in the Wolffian duct and urogenital sinus is regionally restricted and temporally regulated. The domain of expression is defined anteriorly by the caudal epididymis and extends posteriorly to the prostatic anlagen of the urogenital sinus. Expression was maximal at E18 and down-regulated postnatally, well before accessory sex organ morphogenesis is completed. Expression in the prostatic anlagen of the urogenital sinus cultured in vitro does not depend upon the presence of testosterone. Loss of Hoxa-10 function is associated with diminished stromal clefting of the seminal vesicles and decreased size and branching of the coagulating gland. The ductal architecture of the coagulating gland was altered in approximately 30% of mutants examined and suggests a partial posterior morphologic transformation of the coagulating gland. We interpret these data to indicate that Hoxa-10 is expressed in a region specific manner during late gestation and into the perinatal period and that Hoxa-10 is required for normal accessory sex organ development.

Protective effect of FK409, a spontaneous nitric oxide releaser, on ischemic acute renal failure in rats.

The contribution of nitric oxide (NO) to ischemic acute renal failure (ARF) is controversial. In the present study, we investigated the effect of FK409 ((+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexanamide ), a spontaneous NO donor, on ischemic ARF in rats. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal functional parameters such as blood urea nitrogen, plasma creatinine, creatinine clearance, urine flow, urinary osmolality and fractional excretion of sodium were measured to test the effectiveness of the drug. Renal function in untreated ARF rats markedly decreased at 24 hr after reperfusion and thereafter tended to recover gradually. Intravenous bolus injection of FK409 at a dose of 1 mg/kg before the occlusion markedly attenuated the ischemic ARF-induced decreases in renal function, to the same extent as verapamil (1 mg/kg i.v.). The protective effect of FK409, at a dose of 3 mg/kg, was much more potent than that of the lower dose. Histopathological examination of the kidney of untreated ARF rats revealed severe renal damages, such as tubular necrosis, proteinaceous casts in tubuli and medullary congestion. These renal damages were significantly attenuated by treatment with FK409, at each dose given and this attenuation exceeded that seen with verapamil treatment. FK 409 administration led to a dose-dependent increase in NO metabolites concentration in renal venous blood immediately after the reperfusion. These findings suggest that NO has a crucial role in the pathogenesis of ischemic ARF. Spontaneous NO donors may be clinically effective in cases of ischemic ARF.

Auditory evoked off-response: its source distribution is different from that of on-response.

Offset auditory responses were investigated by electroencephalography mainly in the 1970s, but since then no particular attention has been paid to them. Among the studies using magnetoencephalography (MEG) devices there are, to our knowledge, only three studies of the auditory off-response, and no significant variance has ever been observed between the source locations of on- and off-responses elicited from pure tones. We measured auditory evoked magnetic fields (AEFs) to various frequency pure tone stimulation in 5 healthy subjects with a 122-channel helmet-shaped magnetometer, and compared the distributions of the source locations of auditory N100m-Off (magnetic off-response around 100 ms) with those of N100m-On. Their spatial distributions were quite close to each other, and yet they were significantly different.

Ipsilateral dominance of human olfactory activated centers estimated from event-related magnetic fields measured by 122-channel whole-head neuromagnetometer using odorant stimuli synchronized with respirations.

The aim of this study was to measure and analyze olfactory event-related magnetic fields using a whole-cortex biomagnetometer (122-channel SQUID gradiometer). Amyl-acetate gas (approx. 1%) was administered for 300 msec into either the right or left nostril in synchronization with respiration using a mask and an optical fiber sensor. Clear olfactory event-related magnetic fields were asymmetrically obtained on both sides of the forehead in all six subjects. The generators of olfactory magnetic fields were estimated at two regions located fairly asymmetrivally near the bilateral frontal deep areas. The goodness-of-fit was better for the two-dipole model than the one-dipole model in all experiments. In almost all subjects the latency and intensity of ipsilateral olfactory magnetoencephalography (MEG) responses were shorter and larger than those of the contralateral responses, respectively. These results suggest that the olfactory MEG responses on the ipsilateral side are generally larger and more dominant than those on the contralateral side in the human olfactory system.

Inhibition of prostate ductal morphogenesis by retinoic acid.

PURPOSE: To examine the effect of retinoic acid on prostate ductal morphogenesis. MATERIALS AND METHODS: Newborn male Balb/C mice were injected with 25 mg./kg. all-trans retinoic acid or vehicle alone. Animals were sacrificed at 60 days of age and prostate ductal morphology was quantitatively assessed by microdissection. Total prostate DNA was quantitated by DPA assay. RESULTS: The greatest effect was seen in the ventral prostate. Retinoic acid treated animals showed a 20% decrease in mean number of branch-points (p = 0.0006) with a corresponding 13% decrease in duct tips (p = 0.026). The combined ventral and dorsal prostate showed an effect with a 12% decrease in ductal branchpoints (p = 0.048). There was no effect on animal or organ weight and no effect on DNA content within the prostate. There was no difference in the prostate histology of treated and control animals. CONCLUSION: Retinoic acid administration in the newborn period inhibits mouse prostate ductal morphogenesis. This effect appears independent of an inhibition of overall growth.

Micro-magnetic resonance imaging of the inner ear in the guinea pig.

We applied a magnetic resonance microscopy at 7.05 T with a gradient coil unit to image the fine structure of the guinea pig cochlea. First, a three-dimensional MR image of the surface of the cochlea was obtained to select the location of cross-sectional images. Then, cross-sectional images of the basal turn, the second turn and the apical turn of the cochlea were obtained. Based upon the different protein concentrations of the endolymph vs the perilymph, the scala vestibuli, scala tympani and the cochlear duct could be clearly distinguished. This allowed a determination of the location of both the basilar membrane and Reissner's membrane. We raise the possibility that MRM may become useful in the diagnosis of endolymphatic hydrops (Meniere's disease).

Blastomycosis of the epididymis and prostate.

We report a case of blastomycosis presenting as epididymitis and prostatitis. The diagnosis was suggested by pathologic findings in the prostate and epididymis and was further supported by serology. The diagnosis was confirmed by culture and special staining. Long-term cure was accomplished after a 12-month course of oral ketoconazole (400 mg/day). Therapy was monitored by culture and serology. Blastomycosis is an unusual but significant pathogen which occasionally presents with genitourinary tract involvement. Effective diagnostic and oral treatment regimens are now available but are dependent on a high degree of suspicion in cases of chronic prostatitis or epididymitis.

High-resolution MRI of the human cochlea.

The majority of magnetic resonance imaging (MRI) studies in neurotology concern the evaluation of retrocochlear pathologies or temporal bone lesions. Recently, quite a number of investigators have attempted to use imaging diagnosis to obtain a diagnosis and manifest the pathological findings of Meniere's disease. However, there is no evidence that endolymphatic hydrops in patients with Meniere's disease can be identified by imaging techniques. In this study we could depict Reissner's membrane clearer than before with the use of image processing. At the present time, we cannot apply the 2.11T MRI machine to patients under FDA regulation. We believe that MRI diagnoses of endolymphatic hydrops and small lesions of the internal structures of the inner ear will be possible in the near future.

Functional imaging of the human olfactory cortex by magnetic resonance imaging.

Our understanding of the neural mechanism of human olfaction is still equivocal. Several recent reports document that functional magnetic resonance imaging (MRI) has a potential to visualize dynamic brain function in humans without invasion. In the present study, we applied functional MRI with odor stimulation for the purpose of clarifying the localization of olfactory cortices in the human. We obtained a significant increase in cerebral blood flow in the piriform cortex, orbitofrontal cortex, and inferior medial frontal lobe, corresponding to olfactory cortices. These results suggest that, in the near future, precise diagnosis of the patients with olfactory disorders will be possible using functional MRI with odor stimulation.

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.

High-resolution magnetic resonance imaging of the human temporal bone.

A magnetic resonance imaging (MRI) system (Hitachi, Naka, Japan) with a superconductive magnet running at 2.11 T was used to obtain 2-mm-thick slices of fixed, decalcified and celloidin-embedded human temporal bone. The temporal bone was then sectioned and stained for routine histological evaluation. Both the MR images and the histological sections were in the mid-modiolar slice plane, and comparable images and sections were analyzed to confirm the identity of the inner-ear structures visualized on the MR images. The cochlear duct, scala tympani, scala vestibuli and basement membrane of all three cochlear turns were clearly imaged on MRI. In addition, the vestibule and three semicircular ducts were also clearly seen. This study raises the possibility of some day using MRI for the diagnosis of inner-ear diseases.

Effects of ONO-3708, an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on blood vessels.

The pharmacological properties of a novel thromboxane A2/prostaglandin endoperoxide receptor antagonist, ONO-3708, on blood vessels were examined in vitro and in vivo. ONO-3708, 10 microM, inhibited the rabbit aorta contractions induced by thromboxane A2, prostaglandin H2, 11,9-epoxymethano-prostaglandin H2 (U-46619) or prostaglandin F2 alpha without affecting the contractions induced by angiotensin II, serotonin or norepinephrine. ONO-3708, at a concentration of 1 to 100 nM, appeared to be a competitive inhibitor of the contractile responses of the canine basilar artery to 9,11-epithio-11,12-methano-thromboxane A2 (STA2), U-46619 and PGF2 alpha, and a non-competitive inhibitor of the contractile responses to 15-hydroperoxy-eicosatetraenoic acid (15-HPETE). In in vivo studies, ONO-3708 (10 and 100 micrograms/kg per min i.v.) ameliorated the decrease in diameter of the basilar artery induced by the i.v. infusion of STA2 (0.1 microgram/kg per min) in cats. Furthermore, infusion of ONO-3708 (10 and 30 micrograms/kg per min i.v.) prevented the cerebral vasospasm in an experimental subarachnoid hemorrhage model in dogs. These results indicate that ONO-3708 is a potent antagonist of the thromboxane A2/prostaglandin endoperoxide receptor in vitro and in vivo and may be of therapeutic use in preventing cerebral vasospasm.