Ovulation and the movement of the conceptus in the first 35 days of pregnancy in thoroughbred mares.

Data from 937 Thoroughbred mares, when analysed as a single population, showed no significant difference between the activity of the right and left ovaries and the frequency of location of 35-day pregnancies in the right and left uterine horns. Nor were any differences shown when the data were analysed in years or sire groups. However, significantly more ovulations occurred in the right ovary in September and December and in the left ovary in October and November. Whereas the left and right ovulations were equal in lactating and dry mares, there was a highly significant increase in the number of pregnancies in the right uterine horn in dry mares and in the left uterine horn in lactating mares.     

Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) (P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) (P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP₂, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP₃, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 (P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 (P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 (P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.     

X-ray crystallographic study of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transition state.

We have studied the structures of adducts formed between subtilisin BPN' and both benzeneboronic acid and 2-phenylethaneboronic acid by x-ray diffraction techniques. Electron density and difference maps at 2.5 A resolution were computed with phases calculated from a partially refined structure of the native enzyme (R = 0.23 at 2.0 A). Both adducts contain a covalent bond between Ogamma of the catalytic Ser-221 and the inhibitor boron atom. The boron atom is coordinated tetrahedrally, with one of the two additional boronic acid oxygen atoms lying in the "oxyanion hole" and the other at the leaving group site identified in previous studies (ROBERTUS, J.D., Kraut, J. ALDEN, R.A., and BIRKTOFT, J.J. (1972) Biochemistry 11, 4293-4303). Moreover, the previously postulated structure of the tetrahedral intermediate for substrate hydrolysis is isosteric with these boronic acid adducts, which can therefore be considered good models for the transition state complex (KOEHLER, K.K., and LIENHARD, G.E. (1972) Biochemistry 10, 2477-2483). These observations further support the suggestion that an important contribution to stabilization of this transition state complex, relative to both the Michaelis complex and the acyl intermediate, occurs as a consequence of hydrogen bond donation to the substrate carbonyl oxygen atom from the side chain amido group of Asn-155 and from the backbone amido group of Ser-221.     

Advance of mitosis by histone phosphokinase

The previous observation that growth-associated histone kinase (HKG) from Ehrlich ascites cells brings forward mitosis in Physarum polycephalum has been confirmed with more step 1 histone kinase and a more purified (step 2) histone kinase and the statistical significance of the results assessed. The mitosis appears normal in the phase contrast microscope and DNA synthesis is initiated after mitosis as usual. In vitro the growth-associated histone kinase phosphorylates chromatin, the phosphate appearing in F 1 histone. The results are interpreted as providing support for the hypothesis that growth-associated histone kinase controls the initiation of mitosis through F 1 histone phosphorylation and chromosome condensation.     

Pre- and peri-ovulatory distribution of viable spermatozoa in the pig oviduct: a scanning electron microscope study

Using sexually mature animals, the distribution of spermatozoa has been examined at the utero-tubal junction and in the distal and proximal portions of the oviduct isthmus. Mating occurred during early oestrus and, with one exception, specimens were prepared shortly before or after ovulation. Distinct reservoirs of spermatozoa were identified in furrows between the terminal folds of the isthmus, and particularly within the troughs and transverse ridges of this region. The density of spermatozoa diminished steeply from the utero-tubal junction towards the isthmus, especially in the pre-ovulatory specimens. The membranes of most spermatozoa in the isthmus were intact up to the time of ovulation, suggesting that the acrosome reaction is a peri- or post-ovulatory event. Whilst the flagella of spermatozoa in the reservoirs were usually straight or only slightly curved, those on the surface of the epithelial folds were undulating (S-shaped). Specific microenvironments may therefore exist in the distal portion of the isthmus to regulate sperm motility; droplets of secretion were a notable feature in this region. In specimens prepared 24 hr after ovulation, spermatozoa were almost absent from the utero-tubal junction and isthmus. However, denuded eggs were observed in the proximal portion of the isthmus in this animal, and they had spermatozoa associated with the zona pellucida. Arguments are presented for a peri-ovulatory endocrine regulation of sperm redistribution and capacitation.     

2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype

2-Methoxy-3-isobutylpyrazine (MIBP) contributes a bell pepper aroma to many grape cultivars and has a reported aroma threshold of ～2 ng L(-1) in water. The purpose of this study was twofold: (1) develop a procedure using headspace solid phase micro-extraction combined with GC-MS in the selected ion monitoring mode (HS-SPME-GC-MS-SIM) for analysis of MIBP in grape berries, and (2) determine the location of MIBP biosynthesis in grapevines by approach grafting clusters of Vitis vinifera L. cvs Cabernet Sauvignon and Muscat blanc onto each other. The soluble solids and pH of the grape juice/homogenate matrix from different grape berry developmental stages influenced the method precision; therefore, quantification via the method of standard addition was used. Using our developed method, the limit of detection (LOD) and limit of quantitation (LOQ) of MIBP were 0.1 ng L(-1) and 2 ng L(-1), respectively, measured in a model juice and non-MIBP containing Chardonnay juice. Spiked recoveries averaged between 91% and 112% in Cabernet Sauvignon and Pinot noir homogenates and the overall relative standard deviation was less than 10%. The method was used to analyze MIBP in 29 grape cultivars and in fruit from clusters grafted to Cabernet Sauvignon or Muscat vines. Quantifiable levels were found only in Cabernet franc, Cabernet Sauvignon, Merlot, Sauvignon blanc and Semillon, providing information on the genetic connection for the occurrence of MIBP in grapes. No MIBP was detected in the berries of Muscat blanc clusters grafted onto Cabernet Sauvignon vines when sampled at fruit maturity. MIBP was detected in all berries of Cabernet Sauvignon regardless the graft configuration. The data indicate that MIBP or its precursors originate in the berry and its formation depends upon grape genotype.     

Cold and carbon dioxide used as multi-hurdle preservation do not induce appearance of viable but non-culturable Listeria monocytogenes

AIMS: To study whether the exposure to cold (4 degrees C) and carbon dioxide which results in the elongation of Listeria cells, induces a viable but nonculturable (VBNC) state. METHODS AND RESULTS: When cold and CO2 stressed L. monocytogenes were observed under a fluorescence microscope, using the LIVE/DEAD BacLight bacteria viability kit (Molecular Probes, Eugene, OR, USA), the healthy, mildly injured, and the putative VBNC cells accounted for 31.0% of the stressed cell population. By using the selective plate count, 31.4% of the same stressed cell population was found to be healthy and mildly injured (putative VBNC cells not included). If there were VBNC state cells present, we should have observed a significant difference between the above two numbers. In fact, there was no significant difference between the results obtained from those two methods. CONCLUSIONS: There were no VBNC state cells observed in the stressed cell population. We conclude that cold and CO2 do not induce L. monocytogenes to enter a VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold and modified atmospheres are widely used in fresh muscle food and fruit preservation. Whether they would induce L. monocytogenes into a VBNC state is of a great concern for microbial food safety.     

HCV genotype analysis in HCV-HIV-co-infected Puerto Ricans who are injecting drug users: undetermined and mixed infections.

Direct percutaneous exposure is the main route of HCV transmission. In Puerto Rico half of people infected with HIV use illicit drugs. The effects of HCV in the course of HIV infection and vice versa have been extensively studied, but remain highly controversial. This may be due to HCV genetic heterogeneity. Therefore, a complex classification into genotypes has emerged that prompted us to determined how this impacts a population of intravenous drug users (IDUs) co-infected with HIV-1. Using Inno-LiPa II technique, we analyzed samples from 171 HCV-HIV-1-co-infected IDUs and 375 from a general HCV population of unknown HIV or source of infection status. Similar HCV genotype distribution was detected in these populations. HCV genotype 1a was the most frequently in IDUs-co-infected with HIV-1, followed by 1b and 3a. Twenty mixed infections and 5 undetermined genotypes were reported. A reduced HCV viral load was observed in HIV-1 positives with wasting syndrome. Individuals with a high HIV-1 viral load presented a low HCV viral load. There were no correlation between HCV genotypes and AIDS-related event. Patients with genotype 1b showed a higher HCV viral load. Males presented higher HCV viral load than females. Females were predominantly affected by genotype 1a, and men by 1a and 1b. Neither the HCV viral load nor the frequency of genotypes were influenced by the antiretroviral modality. The importance of continuous genotype monitoring is stressed.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.