Characterization of L-type voltage-gated Ca(2+) channel expression and function in developing CA3 pyramidal neurons.

Voltage-gated calcium channels (VGCCs) play a major role during the development of the central nervous system (CNS). Ca(2+) influx via VGCCs regulates axonal growth and neuronal migration as well as synaptic plasticity. Specifically, L-type VGCCs have been well characterized to be involved in the formation and refinement of the connections within the CA3 region of the hippocampus. The majority of the growth, formation, and refinement in the CNS occurs during the third trimester of human pregnancy. An equivalent developmental time period in rodents occurs during the first 2weeks of post-natal life, and the expression pattern of L-type VGCCs during this time period has not been well characterized. In this study, we show that Cav1.2 channels are more highly expressed during this developmental period compared to adolescence (post-natal day 30) and that L-type VGCCs significantly contribute to the overall Ca(2+) currents. These findings suggest that L-type VGCCs are functionally expressed during the crucial developmental period.

The DosR regulon of M. tuberculosis and antibacterial tolerance.

Adaptation of Mycobacterium tuberculosis to an anaerobic dormant state that is tolerant to several antibacterials is mediated largely by a set of highly expressed genes controlled by DosR. A DosR mutant was constructed to investigate whether the DosR regulon is involved in antibacterial tolerance. We demonstrate that induction of the regulon is not required for drug tolerance either in vivo during a mouse infection or in vitro during anaerobic dormancy. Thus, drug tolerance observed in these models is due to other mechanisms such as the bacilli simply being in a non-replicating or low metabolic state. Our data also demonstrate that the DosR regulon is not essential for virulence during chronic murine infection. However, decreased lung pathology was observed in the DosR mutant. We also show that the DosR regulon genes are more highly conserved in environmental mycobacteria, than in pathogenic mycobacteria lacking a latent phase or environmental reservoir. It is possible that the DosR regulon could contribute to drug tolerance in human infections; however, it is not the only mechanism and not the primary mechanism for tolerance during a mouse infection. These data suggest that the regulon evolved not for pathogenesis or drug tolerance but for adaptation to anaerobic conditions in the environment and has been adapted by M. tuberculosis for survival during latent infection.

Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12.

The alternative sigma factor RpoS controls the expression of many stationary-phase genes in Escherichia coli and other bacteria. Though the RpoS regulon is a large, conserved system that is critical for adaptation to nutrient deprivation and other stresses, it remains incompletely characterized. In this study, we have used oligonucleotide arrays to delineate the transcriptome that is controlled by RpoS during entry into stationary phase of cultures growing in rich medium. The expression of known RpoS-dependent genes was confirmed to be regulated by RpoS, thus validating the use of microarrays for expression analysis. The total number of positively regulated stationary-phase genes was found to be greater than 100. More than 45 new genes were identified as positively controlled by RpoS. Surprisingly, a similar number of genes were found to be negatively regulated by RpoS, and these included almost all genes required for flagellum biosynthesis, genes encoding enzymes of the TCA cycle, and a physically contiguous group of genes located in the Rac prophage region. Negative regulation by RpoS is thus much more extensive than has previously been recognized, and is likely to be an important contributing factor to the competitive growth advantage of rpoS mutants reported in previous studies.

Opioid receptor regulation of muscarinic acetylcholine receptor-mediated synaptic responses in the hippocampus.

A common feature of many synapses is their regulation by neurotransmitters other than those released from the presynaptic terminal. This aspect of synaptic transmission is often mediated by activation of G protein coupled receptors (GPCRs) and has been most extensively studied at amino acid-mediated synapses where ligand gated receptors mediate the postsynaptic signal. Here we have investigated how opioid receptors modulate synaptic transmission mediated by muscarinic acetylcholine receptors (mAChRs) in hippocampal CA1 pyramidal neurones. Using a cocktail of glutamate and gamma-amino-butyric acid (GABA) receptor antagonists a slow pirenzepine-sensitive excitatory postsynaptic potential (EPSP(M)) that was associated with a small increase in cell input resistance could be evoked in isolation. This response was enhanced by the acetylcholine (ACh) esterase inhibitor physostigmine (1 microM) and depressed by the vesicular ACh transport inhibitor vesamicol (50 microM). The mu-opioid receptor agonists DAMGO (1-5 microM) and etonitazene (100 nM), but not the delta- and kappa-opioid receptor selective agonists DTLET (1 microM) and U-50488 (1 microM), potentiated this EPSP(M) (up to 327%) without affecting cell membrane potential or input resistance; an effect that was totally reversed by naloxone (5 microM). In contrast, postsynaptic depolarizations and increases in cell input resistance evoked by carbachol (3 microM) were unaffected by DAMGO (1-5 microM) but were abolished by atropine (1 microM). Taken together these data provide good evidence for a mu-opioid receptor-mediated presynaptic enhancement of mAChR-mediated EPSPs in hippocampal CA1 pyramidal neurones.

Indications for pelvic lymphadenectomy in prostate cancer.

Clearly, pelvic lymphadenectomy can provide important staging information in the management of prostate cancer, but this benefit is counterbalanced by a modest increase in morbidity and the significant cost of the procedure. It is difficult to provide universal recommendations concerning the indications for pelvic lymphadenectomy. Part of the problem lies in the fact that urologists perform pelvic lymphadenectomy for several different reasons. Some surgeons perform pelvic lymphadenectomy to better counsel patients after radical prostatectomy about their risk for disease progression and for planning adjuvant radiotherapy or hormonal therapy. For these surgeons, preoperative clinical staging parameters do not exclude patients from pelvic lymphadenectomy, and frozen section analysis intraoperatively provides no useful information. Alternatively, the staging information from pelvic lymphadenectomy can be used to justify cancellation of the subsequent prostatectomy should regional spread of prostate cancer be identified, sparing the patient the morbidity of an unnecessary radical prostatectomy. With this approach, despite the false-negative rate of up to 30%, the expense of frozen section analysis seems justified. For this second group of surgeons, the problem becomes balancing the modest morbidity and cost of pelvic lymphadenectomy against the probability that nodal spread of prostate cancer will be missed if the procedure is omitted. The authors consider a greater than 4% risk for missing regional disease to be unacceptable in this setting. Following this assumption, Table 3 outlines parameters for clinical stage, Gleason score, and preoperative PSA within which pelvic lymphadenectomy is indicated. These recommendations are based on [table: see text] predictions from the Partin nomogram, which has been validated using a series of over 4000 patients. For the large number of patients with clinical T1c disease and a preoperative PSA less than 10 ng/mL, bilateral pelvic lymphadenectomy is indicated only if prostate biopsy identifies tumor of Gleason grade 4 or higher. For lower-grade tumors in this patient population, the risk for nodal metastasis was less than 5% in the Johns Hopkins and Mayo Clinic series of over 5800 patients with prostate cancer. For a large pool of patients, the several thousand dollar cost of pelvic lymphadenectomy and the risk for injury to the obturator nerves and vessels, the formation of lymphoceles, and chronic genital edema can be eliminated with low risk. A nomogram-based approach provides only a starting point for a decision analysis framework to determine whether the surgeon should perform lymphadenectomy at the time of radical prostatectomy because current nomograms predict only lymph node positivity. In a decision analysis framework, some patient and physician value is derived from a negative lymphadenectomy. Moreover, the morbidity associated with pelvic lymphadenectomy and the potential inconvenience associated with treating such morbidity also would be factored into the decision. Consequently, a decision analysis framework that takes into account prognostic value, costs, morbidity, and health state uses ultimately will provide the most informative method for determining when pelvic lymphadenectomy is indicated in patients with prostate cancer.

Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by GABA(B) receptors in the rat hippocampus.

1. Both GABA(B) and muscarinic acetylcholine receptors (mAChRs) influence hippocampal-dependent mnemonic processing. Here the possibility of a direct interaction between GABA(B) receptors and mAChR-mediated synaptic responses has been studied using intracellular recording in rat hippocampal slices. 2. The GABA(B) receptor agonist (-)-baclofen (5-10 microM) depressed an atropine-sensitive slow EPSP (EPSP(M)) and occluded the GABA(B)-receptor-mediated IPSP (IPSP(B)) which preceded it. These inhibitory effects were accompanied by postsynaptic hyperpolarization (9 +/- 2 mV) and a reduction in cell input resistance (12 +/- 3 %). 3. The selective GABA(B) receptor antagonist CGP 55845A (1 microM) fully reversed the depressant effects of (-)-baclofen (5-10 microM) such that in the combined presence of (-)-baclofen and CGP 55845A the EPSP(M) was 134 +/- 21 % of control. 4. (-)-Baclofen (5-10 microM) caused a small (28 +/- 11 %) inhibition of carbachol-induced (3.0 microM) postsynaptic depolarizations and increases in input resistance. 5. CGP 55845A (1 microM) alone caused an increase in the amplitude of the EPSP(M) (253 +/- 74 % of control) and blocked the IPSP(B) that preceded it. 6. In contrast, the selective GABA uptake inhibitor NNC 05-0711 (10 microM) increased the amplitude of the IPSP(B) by 141 +/- 38 % and depressed the amplitude of the EPSP(M) by 58 +/- 10 %. This inhibition was abolished by CGP 55845A (1 microM). 7. Taken together these data provide good evidence that synaptically released GABA activates GABA(B) receptors that inhibit mAChR-mediated EPSPs in hippocampal CA1 pyramidal neurones. The mechanism of inhibition may involve both pre- and postsynaptic elements.

Age-related impairment of synaptic transmission but normal long-term potentiation in transgenic mice that overexpress the human APP695SWE mutant form of amyloid precursor protein.

We have studied synaptic function in a transgenic mouse strain relevant to Alzheimer's disease (AD), overexpressing the 695 amino acid isoform of human amyloid precursor protein with K670N and M671L mutations (APP(695)SWE mice), which is associated with early-onset familial AD. Aged-transgenic mice had substantially elevated levels of Abeta (up to 22 micromol/gm) and displayed characteristic Abeta plaques. Hippocampal slices from 12-month-old APP(695)SWE transgenic animals displayed reduced levels of synaptic transmission in the CA1 region when compared with wild-type littermate controls. Inclusion of the ionotropic glutamate receptor antagonist kynurenate during preparation of brain slices abolished this deficit. At 18 months of age, a selective deficit in basal synaptic transmission was observed in the CA1 region despite treatment with kynurenate. Paired-pulse facilitation and long-term potentiation (LTP) were normal in APP(695)SWE transgenic mice at both 12 and 18 months of age. Thus, although aged APP(695)SWE transgenic mice have greatly elevated levels of Abeta protein, increased numbers of plaques, and reduced basal synaptic transmission, LTP can still be induced and expressed normally. We conclude that increased susceptibility to excitotoxicity rather than a specific effect on LTP is the primary cause of cognitive deficits in APP(695)SWE mice.

Codon bias and base composition are poor indicators of horizontally transferred genes.

Horizontal gene transfer is now recognized as an important mechanism of evolution. Several methods to detect horizontally transferred genes have been suggested. These methods are based on either nucleotide composition or the failure to find a similar gene in closely related species. Genes that evolve vertically between closely related species can be divided into those that retain homologous chromosomal positions (positional orthologs) and those that do not. By comparing open reading frames in the Escherichia coli and Salmonella typhi genomes, we identified 2,728 positional orthologs since these species split 100 MYA. A group of 1,144 novel E. coli genes were unusually diverged from their S. typhi counterparts. These novel genes included those that had been horizontally transferred into E. coli, as well as members of gene pairs that had been rearranged or deleted. Positional orthologs were used to investigate compositional methods of identifying horizontally transferred genes. A large number of E. coli genes with normal nucleotide composition have no apparent ortholog in S. typhi, and many genes of atypical composition do, in fact, have positional orthologs. A phylogenetic approach was employed to confirm selected examples of horizontal transmission among the novel groups of genes. Our analysis of 80 E. coli genes determined that a number of genes previously classified as horizontally transferred based on base composition and codon bias were native, and genes previously classified as native appeared to be horizontally transferred. Hence, atypical nucleotide composition alone is not a reliable indicator of horizontal transmission.

Reduced long-term potentiation in hippocampal slices prepared using sucrose-based artificial cerebrospinal fluid.

Sucrose-based artificial cerebrospinal fluid (aCSF) is sometimes used to prepare brain slices for in vitro electrophysiological experiments. This study compared the effect of preparing brain slices using chilled sucrose-based aCSF versus the conventional method using chilled aCSF on hippocampal synaptic plasticity. Brain slices from each treatment group were transferred to normal aCSF before electrophysiological recordings were made. The stimulus-response relationship of field excitatory postsynaptic potentials (fEPSPs) in the CA1 region was indistinguishable between the two treatment groups. However, the amount of LTP induced by either a θ-burst (four stimuli at 100 Hz repeated ten times at 200 ms intervals) or tetanic stimulation (100 Hz for 1 s) was significantly reduced in slices that had been prepared using sucrose-based aCSF. This was associated with reduced facilitation of the fEPSPs during the high frequency stimulus, reduced post-tetanic potentiation and short-term potentiation. In sucrose-cut slices the fEPSPs were slightly shorter in duration (29%, P<0.01), and during paired-pulse stimulation the broadening of the second fEPSP was enhanced. The LTP deficit in sucrose-cut slices was reversed by blocking GABA(A) receptor function with picrotoxin. These data suggest that the use of sucrose based aCSF better preserves GABA-mediated synaptic transmission, which limits the induction of LTP in hippocampal brain slices.

Expression of bone morphogenetic protein receptors type-IA, -IB and -II correlates with tumor grade in human prostate cancer tissues.

Bone morphogenetic proteins (BMPs) are potential regulators of prostate cancer cell growth and metastasis that signal through an interaction with BMP membrane receptors (BMPRs) type I and type II. In the present study, Western blot and immunohistochemical analysis of BMPRs were carried out in benign and malignant human prostate tissues to explain the loss of BMP response in human prostate cancer cells. The results demonstrated that the benign prostate specimens expressed high levels of all three BMPRs. In normal prostate, BMPRs were localized predominantly to epithelial cells. Among prostate cancer specimens, well-differentiated cancers were positive for the expression of BMPR-II, BMPR-IA, and BMPR-IB, for the most part. In contrast, only 1 of 10 poorly differentiated prostate cancer cases was positive for each of the three BMPRs (P < 0.005 for all three receptors). Taken together, these results indicate that human prostate cancer cells frequently exhibit loss of expression of BMPRs and suggest that loss of BMPRs may play an important role during the progression of prostate cancer.

Similar levels of long-term potentiation in amyloid precursor protein -null and wild-type mice in the CA1 region of picrotoxin treated slices.

Although mutations in amyloid precursor protein (APP) are known to be involved in the development of Alzheimer's disease in some individuals, the role of this protein in normal brain function is poorly understood. We have reported previously that in APP-null mice long-term potentiation (LTP) in the CA1 region of the hippocampus is present but its magnitude is reduced compared to wild-type littermate controls. In the present study, we have confirmed this deficit using a different theta burst induction protocol. Significantly, however, we find that this deficit is no longer apparent when LTP experiments are performed following blockade of gamma-aminobutyric acid(A) receptors. These results suggest that the LTP process per se is not altered by the absence of APP. The deficit may therefore be an indirect consequence of other changes in the hippocampus that occur in the APP-null animal.

Regulation of depolarizing GABA(A) receptor-mediated synaptic potentials by synaptic activation of GABA(B) autoreceptors in the rat hippocampus.

The role of GABA(B) autoreceptors in the regulation of GABA(A) and GABA(B) receptor-mediated inhibitory post-synaptic potentials (IPSPs) during repetitive synaptic activation has been established. In the present study the role of these receptors in the regulation of depolarising GABA(A) receptor-mediated synaptic potentials (DPSP(A)s) in the CA1 region of the hippocampus is documented. Following blockade of AMPA and NMDA receptor-mediated synaptic excitation, DPSP(A)s could be evoked by a single stimulus. The size of this response was enhanced by increasing the stimulus number (1-10 shocks) or stimulus frequency (10-100 Hz). Conversely, the amplitude of the DPSP(A) was dramatically reduced by a priming pulse (single shock) or priming burst (four shocks) delivered 200 ms beforehand. This activity-dependent depression was eliminated by the GABA(B) receptor antagonist CGP 35348 (1 mM). As such, GABA(B) autoreceptor-mediated regulation of DPSP(A)s prevented a pronounced, potentially epileptogenic, DPSP(A) from occurring during theta burst stimulation. Thus, during repetitive stimulation, activation of GABA(B) autoreceptors not only enables a transient reduction in GABA(A) receptor-mediated synaptic inhibition sufficient to enable NMDA receptor-dependent synaptic plasticity [Davies, C.H., Collingridge, G.L., 1996. J. Physiol. 496.2, 451-470] but also prevents the development of a potentially pathogenic depolarising GABA-mediated synaptic potential.

Race is not an independent predictor of positive surgical margins after radical prostatectomy.

OBJECTIVES: To determine whether race is an independent predictor of positive surgical margins in patients who undergo radical prostatectomy. METHODS: Radical prostatectomies were performed on 750 patients at five Veterans Affairs hospitals: Shreveport, Louisiana (n = 451), Houston, Texas (n = 92), Jackson, Mississippi (n = 83), New Orleans, Louisiana (n = 69), and Little Rock, Arkansas (n = 55). All men who did not receive neoadjuvant hormonal therapy and for whom complete follow-up data were available were included in the analysis (of 607, 260 were African-American and 347 were white). Multiple logistic regression analysis was used to determine the significance of race as an independent predictor of surgical margin status after radical prostatectomy for clinically localized prostate cancer. RESULTS: After controlling for clinical stage, Gleason grade, and preoperative prostate-specific antigen (PSA), multivariable logistic regression analysis revealed that race was not an independent predictor of positive surgical margins (P = 0.9). Of the variables evaluated, both preoperative PSA (P = 0.0005) and biopsy Gleason grade (P = 0.047) were significant predictors of an increased risk of a positive surgical margin. CONCLUSIONS: Positive surgical margins are a widely accepted surrogate marker of increased biologic potential in patients with prostate cancer. In our study population, race was not an independent predictor of surgical margin status. Coupled with observations that survival is not related to race, this finding suggests that the biology of prostate cancer in African-American and white men is similar and that observed racial differences more likely are due to ethnic factors that influence tumorigenicity.

Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase dependent and is controlled by RpoS, the alternative vegetative sigma factor.

In the absence of oxygen, many bacteria preferentially use nitrate as a terminal electron acceptor for anaerobic respiration. In Escherichia coli, there are two membrane-bound, differentially regulated nitrate reductases. While the physiological basis for this metabolic redundancy is not completely understood, during exponential growth, synthesis of NRA is greatly induced by anaerobiosis plus nitrate, whereas NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. In the course of identifying genes controlled by the stationary phase regulatory factor RpoS (sigmas), we found that the expression of NRZ is induced during entry into stationary phase and highly dependent on this alternative sigma factor. Expression studies, using operon fusions and nitrate reductase assays, revealed that the NRZ operon is controlled mainly at the level of transcription and is induced 10-fold at the onset of stationary phase in rich media. Consistent with previous reports of RpoS expression, the RpoS dependency of NRZ in minimal media was very high (several hundredfold). We also observed a fivefold stationary phase induction of NRZ in an rpoS background, indicating that other regulatory factors, besides RpoS, are probably involved in transcriptional control of NRZ. The RpoS dependence of NRZ expression was confirmed by Northern analyses using RNA extracted from wild-type and rpoS- strains sampled in exponential and stationary phase. In toto, these data indicate that RpoS-mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress-associated conditions.

The loss of expression of transforming growth factor-beta receptors correlates with the histopathologic tumor grade in bladder transitional cell carcinoma patients.

Transforming growth factor-beta (TGF-beta), a pleiotropic growth factor, is a potent inhibitor of cellular proliferation in cells of epithelial origin. Recently, it has been suggested that a loss of sensitivity to TGF-beta through a loss of expression of TGF-beta receptors T beta R-I and T beta R-II--is associated with tumor initiation and progression. Therefore, to investigate the relationship between TGF-beta receptors expression and carcinogenesis of bladder TCC, this study examined the expression of T beta R-I and T beta R-II in 46 bladder TCC patients using immunohistochemistry. Since histopathological grade is a widely accepted marker of prognosis, the results were compared in relation to the three grades of bladder TCC. The results demonstrated that the loss of TGF-beta receptors expression is associated with increasing histopathological grades of bladder TCC. Specifically, both T beta R-I and T beta R-II were readily detected in all 10 normal bladder mucosa specimens. Likewise, all 6 specimens of grade I TCC samples expressed high levels of both TGF-beta receptors. However, among grade II TCC samples, T beta R-I and T beta R-II were detected in 78% and 89%, respectively: among grade III TCC samples, T beta R-I and T beta R-II were detected in 45% and 41%, respectively. These results suggested that loss of sensitivity to TGF-beta may play a role in the progression of TCC from low to high grade disease.

Mechanisms contributing to the deficits in hippocampal synaptic plasticity in mice lacking amyloid precursor protein.

Abnormal processing of amyloid precursor protein (APP), in particular the generation of beta-amyloid (Abeta) peptides, has been implicated in the pathogenesis of Alzheimer's disease. This study examined the consequences of deleting the APP gene on hippocampal synaptic plasticity, and upon the biophysical properties of morphologically identified neurones in APP-null mice. The hippocampus of APP-null mice had a characteristic increase in gliosis throughout the CA1 region and a disruption of staining for the dendritic marker MAP2 and the presynaptic marker synaptophysin. The disruption of MAP2 staining was associated with a significant reduction in overall dendritic length and projection depth of biocytin labeled CA1 neurones. In two groups of APP-null mice that were examined at 8-12 months, and 20-24 months of age, there was an impairment in the formation of long-term potentiation (LTP) in the CA1 region compared to isogenic age matched controls. This LTP deficit was not associated with an alteration in the amplitude of EPSPs at low stimulus frequencies (0.033 Hz) or facilitation during a 100 Hz stimulus train, but was associated with a reduction in post-tetanic potentiation. Paired-pulse depression of GABA-mediated inhibitory post-synaptic currents was also attenuated in APP-null mice. These data demonstrate that the impaired synaptic plasticity in APP deficient mice is associated with abnormal neuronal morphology and synaptic function within the hippocampus.

Genotype and diet effects in lean and obese Zucker rats fed either safflower or coconut oil diets.

Previously we reported that suckling lean heterozygous (FA/fa) Zucker rats had a number of adipose tissue measurements intermediate between those of homozygous lean (FA/FA) and obese (fa/fa) rats. However, in young adult male rats maintained on a low-fat diet, these differences were no longer apparent (i.e., values for the two lean genotypes were similar). In the present study we determined whether the heterozygous effect of the "fa" gene was dependent on the consumption of a high-fat diet. Mother rats were fed high-fat diets containing either safflower (SOD) or coconut (COD) oil throughout mating and lactation. Homozygous lean male and female rats were bred, as well as obese male and lean heterozygous female rats. Suckling rats were studied at 17 days of age. Additional male rats were maintained on the same diet as their mothers until 11-12 weeks of age. Obese suckling rats had higher body weights than lean pups. Inguinal fat pad weights and pad-to-body weight ratios followed the pattern of obese greater than lean (FA/fa) pups that were greater than lean (FA/FA) pups. A similar relationship was found for adipose tissue lipogenic enzyme activities. At 11-12 weeks of age, measurements followed the general pattern of obese rats having greater values than lean rats (i.e., FA/fa = FA/FA). SOD-fa/fa rats had higher hepatic lipogenic enzyme activities than COD-fa/fa rats. In addition, SOD rats had higher fat cell numbers than COD rats. These results suggest that specific fatty acids can alter adipocyte proliferation and/or differentiation in vivo. In addition, there appears to be a defect of fatty acid regulation in livers of genetically obese rats. The heterozygous effect of the "fa" gene in suckling Zucker rats was confirmed. However, high-fat feeding did not result in a heterozygous effect in young adult lean male rats. We will next evaluate the role of sex on this effect.