录象机在生物组织连续切片的计算机三维重建系统中的应用

录象机(Video Tape Recorder)作为输入、输出设备,应用于“生物组织连续切片的计算机三维重建系统”中,其效果是令人满意的.由于研制的“同步再生”单元(Sync RecoveryUnit)有效地消除了静放噪声,从而使Cromemco微型计算机的图象输入接口SDD(Super DazzlerDigitizer)能够稳定地逐帧采集录象机输出的静止图象.录制并编辑计算机三维重建后的生物组织显微结构的空间旋转视图,使其在录象机的监视器上显示得更生动、逼真,更有体视感;还可脱离主机在任何场合演示重建结果.本文还就录象机在生物医学序列图象分析中的应用前景进行了讨论.     

Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes

The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P<0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P<0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P<0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P<0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.     

Detection of two TaqI polymorphisms in the VTR region of the human HRAS1 oncogene

Restriction enzyme analysis of the variable tandem repetition (VTR) region of the human HRAS1 oncogene revealed two TaqI polymorphisms. The first polymorphism overlaps that detected with MspI/HpaII restriction enzymes and is due to a variable number of repeat units which form the VTR region at the 3' end of the oncogene. The second polymorphism appears to be due to two new TaqI restriction sites created within the VTR region itself. This novel polymorphism was detected in 26 of 145 human DNA samples and was found to segregate in a Mendelian fashion.     

Allele-specific methylation of the human c-Ha-ras-1 gene

The methylation status of individual c-Ha-ras-1 alleles in human cells was measured by Hpall/Mspl analysis of a polymorphic (VTR) region. The gene was extensively methylated in leukocytes and sperm, with the former exhibiting a highly specific methylation pattern. Individual ras alleles were differentially methylated at the VTR region in fetal fibroblasts and immortal cell lines. A new polymorphism in the 5'-flanking region of the gene was also detected. The presence or absence of an Xhol site showed a striking and complete concordance with the length of the VTR region and suggested that the site had been lost by a previous allele-specific mutation.     

Minisatellite allele diversification: the origin of rare alleles at the HRAS1 locus.

Three genetic markers within the promoter-exon 1 region of the HRAS1 locus have been employed to investigate lineage relationships among alleles of the highly polymorphic variable tandem repeat (VTR) immediately downstream of the HRAS1 gene. These markers were in absolute linkage disequilibrium with the HRAS1 VTR, allowing the assignment of unique upstream haplotypes to each of the four common VTR alleles. Analysis of 17 rare alleles revealed a stratification of allele fragment size and upstream haplotype in which each rare VTR allele possessed the markers characteristic of the common allele nearest in size. Therefore, hyperallelism emanated from the four common alleles in a defined fashion, the size of a rare allele specifying its origin. As discussed below, this result implies that unequal crossing-over between homologues is unlikely to be the predominant mechanism for generating new VTR alleles at this minisatellite locus.     

Virtual structural analysis of tibial fracture healing from low-dose clinical CT scans

Quantitative assessment of bone fracture healing remains a significant challenge in orthopaedic trauma research. Accordingly, we developed a new technique for assessing bone healing using virtual mechano-structural analysis of computed tomography (CT) scans. CT scans from 19 fractured human tibiae at 12 weeks after surgery were segmented and prepared for finite element analysis (FEA). Boundary conditions were applied to the models to simulate a torsion test that is commonly used to access the structural integrity of long bones in animal models of fracture healing. The output of each model was the virtual torsional rigidity (VTR) of the healing zone, normalized to the torsional rigidity of each patient’s virtually reconstructed tibia. This provided a structural measure to track the percentage of healing each patient had undergone. Callus morphometric measurements were also collected from the CT scans. Results showed that at 12 weeks post-op, more than 75% of patients achieved a normalized VTR (torsional rigidity relative to uninjured bone) of 85% or above. The predicted intact torsional rigidities compared well with published cadaveric data. Across all patients, callus volume and density were weakly and non-significantly correlated with normalized VTR and time to clinical union. Conversely, normalized VTR was significantly correlated with time to union (R² = 0.383, p = 0.005). This suggests that fracture scoring methods based on the visual appearance of callus may not accurately predict mechanical integrity. The image-based structural analysis presented here may be a useful technique for assessment of bone healing in orthopaedic trauma research.     

Time Course and Localization of the Effects of Estrogen on Glutamic Acid Decarboxylase Activity1

Abstract: Two approaches were used in an attempt to characterize the effect of estrogen on glutamic acid decarboxylase (GAD) [EC 4.1.1.15] activity in ovariectomized rats. In the first experiment, estradiol-17β (E₂) was unilaterally implanted in one of five different brain areas. After 3 days of estrogen exposure, the animals were sacrificed, and GAD activity in the substantia nigra (SN) and ventral tegmental region (VTR) was measured. Estrogen implanted into the preoptic area and the ventromedial nucleus was ineffective, as were implants of cholesterol, regardless of implant site. However, GAD activity was decreased in the SN when E₂ was implanted into the caudate nucleus or amygdala and in the VTR when implanted into the nucleus accumbens septi. Furthermore, this decrease in GAD activity occurred only in the implanted side. In the second experiment, the time course of changes in GAD activity was measured in ovariectomized rats given a single systemic injection of either 8μg estradiol benzoate (EB) or oil. Rats were sacrificed at 0, 12, 29, or 53 h postinjection. It was found that GAD activity in the SN was maximally suppressed 29 h after EB, whereas decreased GAD activity in the VTR was apparent 12 h after EB but had returned to normal by 29 h. Oil injections had no significant effect on GAD activity. These results suggest that there may be two separate and distinct γ-aminobutyric acid pathways, which are differentially responsive to estrogen.     

Human restriction fragment length polymorphisms and cancer risk assessment

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.     

Attempts to determine the status of electroshock-induced attack in male laboratory mice

An attempt was made using a combination of simple experimental manipulations and videotape recorder (VTR) analysis of bite targets employed to determine whether electroshock-induced attack on anosmic opponents in laboratory mice was an offensive or a defensive behaviour. VTR analysis suggested that ventral surface biting was more evident in this form of attack than in social conflict. Individually- and group-housed males showed similar levels of fighting on exposure to electroshock, but dominant males from pairs showed greater attack than their subordinate partners. Zinc sulphate-induced anosmia, 36 h of food deprivation, castration and lithium chloride treatment reduced electroshock-induced attack. Although significant changes were not obtained, there was some evidence that acute treatment with dexamethasone or ACTH augmented this behaviour. The direction of these changes is similar to that seen with social conflict, and it is suggested that electroshock-induced attack in the mouse (unlike the rat) is largely an aggressive offensive behaviour. The high incidence of ventral surface biting may be a consequence of the upright postures assumed on subjecting the animals to electroshock.     

VTR expression cassettes for engineering conditional phenotypes in Pseudomonas: activity of the Pu promoter of the TOL plasmid under limiting concentrations of the XylR activator protein

A simplified procedure to construct recombinant Pseudomonas putida (Pp) and related bacteria, which transcribe conditionally specific genes inserted into their chromosome in response to lac inducers such as IPTG, has been developed. The method is based on the so-called VTR expression cassettes. These are three small (1.98-kb) DNA segments engineered as NotI restriction fragments that include a lacI^q gene along with the hybrid trp/lac promoter, Ptrc, followed by an optimised translation initiation region with a leading ATG and a multiple cloning site in each of the three reading frames. This arrangement allows the chromosomal insertion of the conditionally expressed genes of interest through its transfer to any of the mini-Tn5 transposon vectors available. VTR cassettes permit construction of specialized strains that are instrumental to address, by genetic means, otherwise intractable regulatory problems observed in biodegradative pathways of Pp. In this context, the VTR system was employed to examine the effect of the intracellular concentration of XylR, the main regulator of the TOL (toluene biodegradation) plasmid pWWO, on the exponential silencing of the promoter of the upper operon, Pu. Increasing concentrations of XylR resulted in more intense induction of the system that, however, remained silent during fast cell growth regardless of activator levels.     

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.     

Blood velocity measurement in human conjunctival vessels

The bulbar conjunctiva is one of the few areas in which blood flow in the peripheral vasculature can be directly and noninvasively observed in the human. Although extensive literature exists describing morphological changes which correlate with a variety of systemic diseases in this vasculature, little quantitative data is available on hemodynamics in either normal or abnormal states. The hemodynamic data available are primarily subjective assessments of "low flow." Approaches to place the subjective assessment on more quantitative grounds have usually been based on photographic techniques that have intrinsic inadequacies. The objective of the work reported here was to develop a system capable of providing sequential blood velocity data potentially useful for providing quantitative information on blood flow and its change in the microvessels of the human conjunctiva. The method that has evolved uses a standard Zeiss slit-lamp to image a subject's conjunctival vessels by using a 1-inch Newvicon TV camera with an electronic magnification of 2x. The video image is simultaneously recorded on a video tape recorder (VTR) to an overall system magnification of approximately 4 microm/raster line. The data acquisition phase requires approximately 5 minutes of patient time, whereas the actual determination of blood velocity in individual vessels is done offline through a modification of the dual-slit videodensimetric method. Two independently controllable video cursors are placed axially over the vessel image with the VTR in the still-frame mode. For each consecutive video field, the position of two reference points on the vessel and the position of each cursor relative to these and to each other are encoded into a computer to track the moving image caused by normal eye movement. The computer then determines new cursor coordinates to ensure a constant position within the vessel. The electrical signals obtained for each cursor site and for each video field are cross-correlated to yield the average blood velocity over the sampled time interval. The system has been calibrated in vitro from 0.2 to 2.5 mm/sec, evaluated in experimental animals, and used to measure blood velocity (0.3 to 1.5 mm/sec) in human conjunctival venules with diameters ranging from 20 to 50 microm. At this writing, blood velocity has been recorded during a period of about 3 months in the same vessel of several postmyocardial infarction patients. Thus, the method appears suitable for determining sequential changes in small vessel blood flow in patients over extended periods of time.     

The human minisatellite consensus at breakpoints of oncogene translocations.

A reexamination of human minisatellite (hypervariable) regions following the cloning and sequencing of the new minisatellite, VTR1.1, revealed that many of these structures possessed a strongly conserved copy of the chi-like octamer, GC[A/T]GG[A/T]GG. In oncogene translocations apparently created by aberrant VDJ recombinase activity, this VTR octamer was often found within a few bases of the breakpoint (p less than 10(-10)). Three bcl2 rearrangements which occurred within 2 bp of one another were located precisely adjacent to this consensus; it defined the 5' border of that oncogene's major breakpoint cluster. Several c-myc translocations also occurred within 2 bp of this sequence. While the appearance of a chi-like element in polymorphic minisatellite sequences is consistent with a role promoting either recombination or replication slippage, the existence of such elements at sites of somatic translocations suggests chi function in site-specific recombination, perhaps as a subsidiary recognition signal in immunoglobulin gene rearrangement. We discuss the implications of these observations for mechanisms by which oncogene translocations and minisatellite sequences are generated.     

Evaluation of antifungal activity of antimycotics by automatic analyzing system

Antifungal activity of several antimycotics has been evaluated using an automatic analyzing system (AAS), which is composed of a specially designed reaction vessel, microscopic observation system, image analyzing system, and computer program for automatic tracing of hypha growth. The agar plate was prepared on the ceiling of the reaction vessel, and spore mass of a fungus (Aspergillus niger) was inoculated onto it. After the preincubation at 28 °C for 24 h the reaction vessel was set on a microscope stage and connected to the liquid flow system. An appropriate hypha was selected for the measurement of growth process during the following steps: first contact with saline for 30 min for the adaptation, the second contact with same saline for 30 min, contact with saline containing an antimycotic substance for 60 min, and contact with flushing saline for 60 min. During a sequence of these steps, the apical tip of a growing hypha displayed on a TV monitor was followed automatically. The dynamic response of hypha to an agent was analyzed by several parameters. Morphological changes of the hypha caused by respective agents were recorded on VTR for further analysis. By using this system, the antifungal activity of antimycotics could be quantitatively determined within several hours.     

Genetic analysis of H-ras intron-1 polymorphic and variable tandem repeat regions in human breast cancer

H-ras is a member of the ras superfamily of genes. This gene encodes for a 21 kDa protein (p21) which is located on the inner surface of the plasma membrane. Ras genes are involved in a wide variety of human tumors, and there is a known correlation between H-ras activation and breast carcinogenesis. H-ras contains a polymorphic region, a repeated hexanucleotide -GGGCCT - located in intron 1 close to the 5' of the gene (HRM region). Three alleles of this region, P1, P2 and P3, have been identified that contain two, three and four repeats of the hexanucleotide, respectively. H-ras possesses a minisatellite DNA of the variable tandem repeat (VTR) which is located 1000 bp downstream of the gene displaying linkage disequilibrium with HRM. The purpose of this study was to estimate the frequency of P1, P2 and P3 in the normal population and in patients with breast cancer. We studied 56 biopsy specimens from patients with breast cancer, 61 normal blood samples, and 30 pairs of normal and tumoral breast tissues for VTR analysis. There was a difference in the distribution of P1, P2 and P3 alleles between normal and breast cancer samples. The frequency of P1 homozygosity was shown to be almost twice as high in women with breast cancer compared to healthy women (72% versus 39%). These results suggest that P1 homozygosity may be considered as a potential risk factor in breast carcinogenesis. In VTR analysis one sample presented a shift in mobility, but no polymorphism in the BstN I pattern of the 28 bp repetition core was observed.     

Orexin A (hypocretin 1) injected into hypothalamic paraventricular nucleus and spontaneous physical activity in rats

In humans, nonexercise activity thermogenesis (NEAT) increases with positive energy balance. The mediator of the interaction between positive energy balance and physical activity is unknown. In this study, we address the hypothesis that orexin A acts in the hypothalamic paraventricular nucleus (PVN) to increase nonfeeding-associated physical activity. PVN-cannulated rats were injected with either orexin A or vehicle during the light and dark cycle. Spontaneous physical activity (SPA) was measured using arrays of infrared activity sensors and night vision videotaped recording (VTR). O(2) consumption and CO(2) production were measured by indirect calorimetry. Feeding behavior was assessed by VTR. Regardless of the time point of injection, orexin A (1 nmol) was associated with dramatic increases in SPA for 2 h after injection (orexin A: 6.27 +/- 1.95 x 10(3) beam break count, n = 24; vehicle: 1.85 +/- 1.13 x 10(3), n = 38). This increase in SPA was accompanied by compatible increase in O(2) consumption. Duration of feeding was increased only when orexin A was injected in the early light phase and accounted for only 3.5 +/- 2.5% of the increased physical activity. In a dose-response experiment, increases in SPA were correlated with dose of orexin A linearly up to 2 nmol. PVN injections of orexin receptor antagonist SB-334867 were associated with decreases in SPA and attenuated the effects of PVN-injected orexin A. Thus orexin A can act in PVN to increase nonfeeding-associated physical activity, suggesting that this neuropeptide might be a mediator of NEAT.     

泽陆蛙和饰纹姬蛙蝌蚪不同热驯化下选择体温和热耐受性

用泽陆蛙（Fejervarya limnocharis）蝌蚪和饰纹姬蛙（Microhyla ornata）蝌蚪做研究模型,检测热驯化（20 、25 和30 C）对选择体温（Tsel）、低温耐受性（CTMin）和高温耐受性（CTMax）的影响。结果显示,两种蝌蚪的Tsel既不受驯化温度的影响,也不存在种间差异;泽陆蛙蝌蚪的CTMin显著小于饰纹姬蛙蝌蚪,而CTMax和VTR则显著大于饰纹姬蛙蝌蚪;CTMin和CTMax随驯化温度的升高而升高,VTR则随驯化温度的升高而减小。研究结果表明,热驯化显著影响两种蝌蚪的CTMin、CTMax和VTR,而对两种蝌蚪的体温调定点无显著影响;这些热生物学特征对两种蝌蚪有效适应环境温度变化、利用资源、减少种间竞争具有重要的生态学意义。     

Automatic antifungal activity analyzing system on the basis of dynamic growth process of a single hypha

A system for the evaluation of antifungal activity of volatile compounds has been developed that is based on dynamic growth of a single hypha. The newly developed system is composed of a reaction vessel under a microscope, automatic stage, charge coupled device (CCD) camera, TV monitor, video tape recorder (VTR), and a microcomputer. A fungus was inoculated in the reaction vessel containing agar medium and then was treated with an antifungal reagent in the gas phase either in batch or flow reaction manner. The apex of a growing hypha displayed on a TV monitor was followed automatically. From the ratio of the growth rate under exposure of a reagent (U_EXPO) to the growth rate before the exposure (U_PRE), the antifungal activity was expressed quantitatively.     

Hormonal regulation of vasotocin receptor mRNA in a seasonally breeding songbird

Behaviors associated with breeding are seasonally modulated in a variety of species. These changes in behavior are mediated by sex steroids, levels of which likewise vary with season. The effects of androgens on behaviors associated with breeding may in turn be partly mediated by the nonapeptides vasopressin (VP) and oxytocin (OT) in mammals, and vasotocin (VT) in birds. The effects of testosterone (T) on production of these neuropeptides have been well-studied; however, the regulation of VT receptors by T is not well understood. In this study, we investigated steroid-dependent regulation of VT receptor (VTR) mRNA in a seasonally breeding songbird, the white-throated sparrow (Zonotrichia albicollis). We focused on VTR subtypes that have been most strongly implicated in social behavior: V1a and oxytocin-like receptor (OTR). Using in situ hybridization, we show that T-treatment of non-breeding males altered V1a and OTR mRNA expression in several regions associated with seasonal reproductive behaviors. For example, T-treatment increased V1a mRNA expression in the medial preoptic area, bed nucleus of the stria terminalis, and ventromedial hypothalamus. T-treatment also affected both V1a and OTR mRNA expression in nuclei of the song system; some of these effects depended on the presence or absence of a chromosomal rearrangement that affects singing behavior, plasma T, and VT immunolabeling in this species. Overall, our results strengthen evidence that VT helps mediate the behavioral effects of T in songbirds, and suggest that the chromosomal rearrangement in this species may affect the sensitivity of the VT system to seasonal changes in T.     

The “EE-OO machine”—An apparatus which controls a videotape recorder by detecting specific vocalisations

This paper describes a device for detecting a specific vocalisation inMacaca fascicularis. It is used to control a videotape recorder and therefore to record behaviour following the vocalisation. It is activated if two pre-set frequencies occur in a particular order and within a specified time interval. The running time of the VTR is also pre-set and can be varied within wide limits. The circuitry is relatively simple, being based on readily available integrated circuits. In use the machine has proved to be sensitive and to show a high level of specificity.