Matrix Metalloproteinases -14, -9 and -2 are Localized to the Podosome and Involved in Podosome Development in the A7r5 Smooth Muscle Cell.

AIM: The purpose of the study was to localize matrix metalloproteinase (MMP)-14, -9, and -2 in the A7r5 smooth muscle cell and to understand the interaction between these MMPs and the cytoskeleton. This interaction was observed under non-stimulating and phorbol 12, 13-dibutyrate (PDBu)-stimulating conditions. METHODS: Confocal microscopy was utilized to define the localizations of MMPs and tissue inhibitor of matrix metalloproteinases (TIMPs) in the A7r5 cell and to determine interaction between MMPs and the cytoskeleton. Under PDBu-stimulating conditions, the presence of MMP active forms and activity by gel zymography was evaluated in the A7r5 cell. Actin and microtubule-polymerization inhibitors were used to evaluate MMP interaction with the cytoskeleton and the cytoskeleton was observed on matrix and within a Type I collagen gel. RESULTS: MMP-14, -9, and -2 were localized to the podosome in the A7r5 smooth muscle cell and interactions were seen with these MMPs and the actin cytoskeleton. PDBu-stimulation induced increases in the protein abundance of the active forms of the MMPs and MMP-2 activity was increased. MMPs also interact with a-actin and not ß-tubulin in the A7r5 cell. Galardin, also known as GM-6001, was shown to inhibit podosome formation and prevented MMP localization to the podosome. This broad spectrum MMP inhibitor also prevented collagen gel contraction and prevented cell adhesion and spreading of A7r5 cells within this collagen matrix. CONCLUSION: MMPs are important in the formation and function of podosomes in the A7r5 smooth muscle cell. MMPs interact with a-actin and not ß-tubulin in the A7r5 cell. Podosomes play an important role in cell migration and understanding the function of podosomes can lead to insights into cancer metastasis and cardiovascular disease.

How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

The objective of this study was to compare the aetiologic yield of standard-of-care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal (GI) Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of three (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (p < 0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for Clostridium difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents.

Behavioral plasticity in honey bees is associated with differences in brain microRNA transcriptome.

Small, non-coding microRNAs (miRNAs) have been implicated in many biological processes, including the development of the nervous system. However, the roles of miRNAs in natural behavioral and neuronal plasticity are not well understood. To help address this we characterized the microRNA transcriptome in the adult worker honey bee head and investigated whether changes in microRNA expression levels in the brain are associated with division of labor among honey bees, a well-established model for socially regulated behavior. We determined that several miRNAs were downregulated in bees that specialize on brood care (nurses) relative to foragers. Additional experiments showed that this downregulation is dependent upon social context; it only occurred when nurse bees were in colonies that also contained foragers. Analyses of conservation patterns of brain-expressed miRNAs across Hymenoptera suggest a role for certain miRNAs in the evolution of the Aculeata, which includes all the eusocial hymenopteran species. Our results support the intriguing hypothesis that miRNAs are important regulators of social behavior at both developmental and evolutionary time scales.

FRET analysis of actin-myosin interaction in contracting rat aortic smooth muscle.

We examined the interaction of smooth muscle myosin with alpha-actin and beta-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from alpha-actin but not beta-actin. Whole-cell FRET analysis confirmed these observations (alpha-actin-myosin -67%, beta-actin-myosin -2%). Time course studies further showed that alpha-actin-myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in alpha-actin-myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (-4%). By comparison, beta-actin-myosin complex was unchanged except during slow force development, in which the association was significantly decreased (-30%). Similar to that of alpha-actin-myosin, Alexa 488 - phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of alpha-actin-myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein-protein associations.

Differential actin isoform reorganization in the contracting A7r5 cell.

In the present study, we investigated the reorganization of alpha- and beta-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (< or =10(-7) mol/L), cells showed an approximately 70% loss in alpha-actin stress fibers with robust transport of this isoform to podosomes. By comparison, beta-actin remained in stress fibers in cells stimulated at low concentrations (< or =10(-7) mol/L) of PDBu. However, at high concentrations (> or =10(-6)mol/L) approximately 50% of cells showed transport of beta-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PDBu-induced (5 x 10(-8) mol/L) loss in alpha-actin stress fibers or reversed podosome formation with re-establishment of alpha-actin stress fibers. By comparison, these inhibitors caused partial loss of beta-actin stress fibers. The results support our earlier conclusion of independent remodeling of alpha- and beta-actin cytoskeletal structure and suggest that the regulation of these structures is different.

Risk factors for low bone mineral density in individuals residing in a facility for the people with intellectual disability.

BACKGROUND: Individuals with intellectual disability (ID) are known to have a high prevalence of both low bone mineral density (BMD) and fractures with significant attendant morbidity. Effective strategies aimed at reducing fractures will be facilitated by the identification of predisposing risk factors. METHODS: Bone mineral density was measured by quantitative ultrasound of the calcaneus performed on 79 women and 132 men residing in a facility for adults with ID. Multiple variable logistic regression analysis was performed to determine the significance of risk factors for low BMD. RESULTS: Mobility impairment consistently appeared to be a significant risk factor for low BMD regardless of age or sex and especially for middle-aged men with profound ID. Further risk was identified for postmenopausal women taking enzyme inducing anticonvulsant medications and middle-aged men who were either smokers or tended to be short. Hispanic followed by Caucasian origin also put middle-aged males at a greater risk than their African-American counterparts. CONCLUSIONS: Specific risk factors for low BMD, some of which have potential for modification, were identified in the study population. Targeted strategies for risk factor reduction may result in a decrease in the high rate of fractures among these individuals.

Sex-selection of human spermatozoa: evolution of current techniques and applications.

Methods claiming to achieve sex selection by sperm sorting have existed for many years. Numerous applications for safe and effective selection procedures exist in current clinical practice, as sex-linked conditions could be theoretically eliminated by use of appropriate sperm for fertilization or insemination. Use of such techniques could also address the need to effect family balancing for some couples. Modern preconception sex-selection methods may be classified into two general types: those that attempt to segregate spermatozoa on the basis of subtle physical or kinetic features, and those that rely on distinctive nuclear characteristics unique either to X- or Y-chromosome bearing sperm. Laboratories providing sperm sexing using the former method have been available for some years, although the associated efficiency and reproducibility are controversial. Sex selection of spermatozoa by chromatin differences has been shown to achieve significant enrichment of X- or Y-chromosome bearing sperm, but clinical experience in humans is limited. The fundamental elements of the two approaches introduced here are reviewed and compared. Selected key historical concepts in sex selection by sperm sorting are outlined, followed by a summary of promising areas for future research.

Mitochondrial inheritance is delayed in Saccharomyces cerevisiae cells lacking the serine/threonine phosphatase PTC1.

In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence. Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell. Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport. PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway. The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton. Moreover, epistasis experiments with ptc1delta and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance. Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell. These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site.

Selective posterior rhizotomy for children with cerebral palsy: a 7-year experience.

Although selective posterior rhizotomy (SPR) was pioneered as early as 1913, only over the past decade has the procedure gained popular use for the treatment of spasticity in cerebral palsy. The medical knowledge base regarding this procedure is expanding, and surgical techniques continue to be revised. We present our 7 years of experience in treating spastic cerebral palsy using SPR. The aspects of preoperative evaluation used by the multidisciplinary team to determine candidacy are outlined. The surgical procedure is detailed with a particular emphasis on the role of intraoperative nerve root stimulation to aid in selection for rootlet sectioning. Historical nerve stimulation protocols are reviewed and compared to our findings over the years. The functional goals are discussed in the context of the postoperative evaluation and therapies. Specific outcome in relation to joint range of motion, self care tasks, and ambulation is reported. The paper outlines a concise overview of our experiences and will assist the clinician in defining a protocol and expectations for SPR.

Paraneoplastic subacute cerebellar degeneration: functional improvement and the role of rehabilitation.

Paraneoplastic syndromes, or the remote effects of cancer on the nervous system, can result in significant functional impairment. One syndrome in particular, paraneoplastic subacute cerebellar degeneration (PSCD), may be severely disabling. Patients with PSCD can experience severe ataxia resulting in an inability to ambulate or perform their activities of daily living. Little has been written about the value of rehabilitation in cases of paraneoplastic syndrome. We report the case of a 51-year-old woman with PSCD who experienced improvements in all functional activities after comprehensive inpatient rehabilitation. She has maintained her improved functional status after discharge; her case is testimony to the value of rehabilitation in paraneoplastic syndrome.

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit.

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, lyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P < 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.05) or HIS-(8.8%, P < 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in lyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

Pregnancy in a non-communicating uterine horn mimicking incarceration with sacculation of a retroflexed uterus.

Incarceration and sacculation of a retroflexed gravid uterus is relatively benign, in contrast with pregnancy in a rudimentary uterine horn which can lead to perforation and hemorrhage. We report a case of pregnancy in an incarcerated sacculated non-communicating rudimentary horn mimicking incarceration with sacculation of a retroflexed gravid uterus. Both physical and sonographic findings were unhelpful in the differential diagnosis. Therefore, decision for laparotomy in such cases should be based on severity of symptoms.

A unique haplotype of the apolipoprotein B-100 allele associated with familial defective apolipoprotein B-100 in a Chinese man discovered during a study of the prevalence of this disorder.

The prevalence of familial defective apolipoprotein (apo) B-100 (FDB) was determined by sampling 5,160 volunteer subjects from among 14,058 eligible employees of a bank in California. The sample was ethnically diverse (44.6% of the population was non-Caucasian). The prevalence of FDB in the study population was 0.08% with a 90% confidence interval of 0.01-0.14%. Four subjects were found to have the apoB 3500 codon mutation by mutagenic polymerase chain reaction, which creates an MspI site at the 3500 codon of normal alleles but not alleles coding for the Arg-->Gln mutation of FDB. Three of these were Caucasian and born in North America. The fourth was a native of China. Haplotype analysis of the affected allele of the Chinese subject using 10 markers described by Ludwig and McCarthy (1990. Am. J. Hum. Genet. 47: 712-720) revealed a unique haplotype that differed from the haplotype of all other subjects with FDB. This unique allele had 30 repeats of a 3' hypervariable element instead of 48 as was found in the allele associated with FDB in other subjects, and in the 3' region there was an EcoRI site that was also not present in the allele most commonly found in association with FDB. We conclude that the prevalence of FDB in our ethnically diverse population is lower than that reported in previous studies of predominantly Caucasian populations and that the Chinese subject represents either an independent mutation or possibly recombination at the 3' end of the apoB gene, an event not previously described.

Hypolipidemic effects of nicotinic acid in patients with familial defective apolipoprotein B-100.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder associated with hypercholesterolemia, in which an amino acid substitution in apolipoprotein B-100 results in low-density lipoprotein (LDL) particles that bind poorly to the LDL receptor and accumulate in plasma. Patients with FDB described to date have been heterozygous for this disorder, and their plasma contains both normal and defective-binding LDL particles. We have evaluated the hypocholesterolemic effects of nicotinic acid (3 g/d) in four patients with FDB, and compared the response to that of nine patients with heterozygous familial hypercholesterolemia (FH). Concentrations of LDL decreased by 24% in patients with FDB and by 14% in patients with FH. These results support the view that drugs which reduce LDL synthesis may be particularly effective in the treatment of patients with FDB.

Influences of in vitro oocyte aging on microfertilization in the mouse with reference to zona hardening.

PURPOSE: Our purpose was to investigate the influence of in vitro oocyte aging on fertilization and subsequent embryonic development following subzonal sperm injection with reference to spontaneous zona hardening in the mouse. RESULTS: First cleavage rate was significantly higher in long vs short (69 vs 41%, P < 0.001). However, significantly higher blastocyst formation and hatching were observed in short than long (50 vs 7%, P < 0.001, and 86 vs 50%, P < 0.001, respectively). No significant differences were found for two pronuclei formation between short and long (30 vs 27%). Sham injection revealed a significantly higher parthenogenetic activation in long than short (19 vs 2%, P < 0.001). Zona digestion required a significantly longer time for long compared to short (trypsin--37 vs 29 min, P < 0.01; pronase--17 vs 14 min, P < 0.001). CONCLUSIONS: Prolonged culture of mature mouse oocytes in vitro alters the zona pellucida, increases the rate of parthenogenetic activation by subzonal sperm injection, and impairs subsequent embryonic development. Zona hardening appears to be an indicator of oocyte aging.

Baseline ovarian cysts do not affect clinical response to controlled ovarian hyperstimulation for in vitro fertilization.

OBJECTIVE: To evaluate the effect of baseline ovarian cysts at the onset of controlled ovarian hyperstimulation for in vitro fertilization (IVF) on cycle outcome. DESIGN, PATIENTS: A review of 82 IVF cycles in 29 women in which each patient served as her own control. The stimulation regimen for each patient remained constant over time. Each woman had at least one cycle in which an ovarian cyst measuring 14 to 53 mm was present at baseline and one cycle in which no such cyst was present. SETTING: The In Vitro Fertilization Program at Yale University School of Medicine. RESULTS: There was no statistically significant difference in cycle cancellation rates, baseline serum estradiol (E2), peak serum E2, number of follicles present at retrieval, number of oocytes retrieved, or fertilization rate between groups. Stimulation regimen, cyst size, and age were unrelated to outcome. The number of cysts present at baseline correlated positively with the number of follicles present at retrieval. CONCLUSION: Baseline ovarian cysts in the setting of a low baseline E2 level do not affect the clinical response to controlled ovarian hyperstimulation in IVF cycles.

Corpus luteum function in successful in vitro fertilization cycles.

The function of the corpus luteum in early pregnancy has been subject to some controversy. The purpose of our study was to determine the life span of the corpus luteum in early pregnancy after successful GnRH-a/hMG stimulation in IVF-ET. The study consisted of a retrospective analysis of patients after 12 successful singleton intrauterine IVF-ET cycles. Serum samples were obtained during early pregnancy beginning 14 days after hCG administration. The levels of 17 alpha-OHP, hCG, P, and E2 were measured in each sample. A significant negative correlation was noted between 17 alpha-OHP and date from hCG. The x-intercept of the regression line allowed estimation of the life span of the corpus luteum to be 72 +/- 25 days. In conclusion, in GnRH-a/hMG-stimulated IVF-ET cycles that result in a singleton pregnancy, the functional life span of the corpus luteum averages 72 days.

Follicular size and steroid secretion of dominant bovine follicles.

Twenty one estrogen-active bovine follicles obtained in the preovulatory period prior to the endogenous LH peak were superfused for 10h. Follicular diameter and superfusate estradiol (E2), testosterone (T) and progesterone (P4) were measured. A close correlation between follicle size and E2 secreted into the superfusate was found (r = 0.77; p less than 0.01). There was no correlation between superfusate T or P4 and follicular diameter. These data indicate that within certain limits the amount of E2 secreted can be predicted by follicle size in an in vitro superfusion system. As follicle size correlates closely with granulosa cell number in estrogen-active follicles, one can speculate that the rise in estrogen secretion is a reflection of an increase in granulosa cell number of the dominant bovine follicle. The application of these in vitro findings to the in vivo situation might help in the interpretation of the quality of ovarian stimulation with hMG/hCG.

A critical assessment of the indications for in-vitro fertilization and embryo transfer.

In-vitro fertilization therapy (IVF-ET) should be considered an important and indispensable component of all comprehensive infertility treatment programmes. It is neither more, nor less important than operative laparoscopy, hysteroscopic diagnosis, a broad-based programme for ovarian stimulation and ovulation induction or even psychological counseling. Clearly, with today's technology, IVF-ET is not a panacea of infertility, but in selected cases it may provide a child where other forms of therapy have failed. However, what sets this therapy apart from all others is that it has heralded a new era in human biology that will not only enable us to treat, but also to understand the problem of infertility.

Ovarian cysts decrease the success of controlled ovarian stimulation and in vitro fertilization.

Forty cystic structures, 16 to 60 mm, of probable ovarian origin were found in 126 cycles during an ultrasound scan on day 3 of a cycle in which in vitro fertilization was planned. The response of these patients to exogenous gonadotropin stimulation, oocyte capture, fertilization, cleavage, and pregnancy was studied. Patients with cystic change had significantly lower peak estradiol (E2) levels. Pregnancy rates were lower, but not significantly so. Those patients with structures 16 to 29 mm had more cycles canceled due to precipitous drops in E2, consistent with an untimely surge of luteinizing hormone. Patients with structures 30 to 60 mm had increased numbers of cycles with poor response to stimulation. The importance of an early follicular phase ultrasound scan is stressed, and the management of ovarian cysts in this patient population is discussed.