Failure of the orally active progestin, Regu-mate, to overcome confinement-induced delayed puberty in gilts

Thirty-three crossbred gilts that were raised in total confinement were randomly allotted to two adjacent pens in a finishing unit at 144.7 +/- .5 days of age and 58.0 +/- 1.7 kg body weight. At approximately 253 days of age, 16 gilts were group fed a daily dose of 20 mg of Regu-mate per gilt for 18 days and 17 control gilts were group fed the same diet without Regu-mate for 18 days. Ovarian morphology was examined on 11 or 12 days after the last feeding of Regu-mate. Based on estrous behavior and ovarian morphology only one Regu-mate gilt displayed an estrus but did not ovulate and only three of the control gilts displayed estrus and ovulated at least once before the start of treatment. Three of the 16 Regu-mate gilts displayed estrus and ovulated 7.3 +/- .3 days after the last feeding and within the same time period the 3 control gilts, which previously displayed estrus, continued to have estrous cycles. One additional control gilt displayed estrus and ovulated 5 days after the last feeding of the control diet. Therefore, the proportion of gilts that displayed estrus and ovulated by the end of the experimental period were similar for the treated (25.0%) and control (23.5%) groups. Based on these results we conclude that treatment of gilts in a state of confinement-induced delayed puberty with 20 mg Regu-mate daily for 18 days failed to result in a synchronized onset of puberty in a significant proportion of gilts.     

SecA: the ubiquitous component of preprotein translocase in prokaryotes

SecA is an obligatory component of the complex hetero-septameric translocase of prokaryotes. It is unique in that it exists as two forms within the holoenzyme; first, as a structural component of the preprotein channel and second, as an ATP-dependent membrane cycling factor facilitating the translocation of a broad class of proteins across the cytoplasmic membrane. While the translocase activity of SecA appears to be functionally conserved, it is not clear whether the mechanisms of regulation of the secA gene are similarly maintained. The recent characterization of an ATP-dependent RNA helicase activity of SecA offers a unique mechanism for SecA to communicate the secretion status of the cell to the appropriate regulatory circuits simply by the unwinding of an appropriate RNA target. Resolution of these two activities through combined biochemical, genetic, and biophysical studies should lead to a better understanding of the role of SecA in bacterial secretion.     

Containment of simian immunodeficiency virus infection: cellular immune responses and protection from rechallenge following transient postinoculation antiretroviral treatment

To better understand the viral and host factors involved in the establishment of persistent productive infection by primate lentiviruses, we varied the time of initiation and duration of postinoculation antiretroviral treatment with tenofovir (9-[2-(R)-(phosphonomethoxy)propyl]adenine) while performing intensive virologic and immunologic monitoring in rhesus macaques, inoculated intravenously with simian immunodeficiency virus SIVsmE660. Postinoculation treatment did not block the initial infection, but we identified treatment regimens that prevented the establishment of persistent productive infection, as judged by the absence of measurable plasma viremia following drug discontinuation. While immune responses were heterogeneous, animals in which treatment resulted in prevention of persistent productive infection showed a higher frequency and higher levels of SIV-specific lymphocyte proliferative responses during the treatment period compared to control animals, despite the absence of either detectable plasma viremia or seroconversion. Animals protected from the initial establishment of persistent productive infection were also relatively or completely protected from subsequent homologous rechallenge. Even postinoculation treatment regimens that did not prevent establishment of persistent infection resulted in downmodulation of the level of plasma viremia following treatment cessation, compared to the viremia seen in untreated control animals, animals treated with regimens known to be ineffective, or the cumulative experience with the natural history of plasma viremia following infection with SIVsmE660. The results suggest that the host may be able to effectively control SIV infection if the initial exposure occurs under favorable conditions of low viral burden and in the absence of ongoing high level cytopathic infection of responding cells. These findings may be particularly important in relation to prospects for control of primate lentiviruses in the settings of both prophylactic and therapeutic vaccination for prevention of AIDS.     

Staphylococcus aureus cap5P Encodes a UDP-N-Acetylglucosamine 2-Epimerase with Functional Redundancy

The serotype 5 capsule gene cluster of Staphylococcus aureus comprises 16 genes (cap5A through cap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that the N-acetylmannosaminuronic acid (ManNAcA) component of the S. aureus serotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded by S. aureus cap5P. Purified Cap5P converted approximately 10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression in S. aureus was unaffected by insertional inactivation of cap5P. Sequence homology searches of the public S. aureus genomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on the S. aureus chromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function in S. aureus was demonstrated by cloning the cap5P homologue from strain Newman and complementing an Escherichia coli rffE mutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of the S. aureus cap5P gene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.     

Defining natural history: assessment of the ability of college students to aid in characterizing clinical progression of Niemann-Pick disease, type C

Niemann-Pick Disease, type C (NPC) is a fatal, neurodegenerative, lysosomal storage disorder. It is a rare disease with broad phenotypic spectrum and variable age of onset. These issues make it difficult to develop a universally accepted clinical outcome measure to assess urgently needed therapies. To this end, clinical investigators have defined emerging, disease severity scales. The average time from initial symptom to diagnosis is approximately 4 years. Further, some patients may not travel to specialized clinical centers even after diagnosis. We were therefore interested in investigating whether appropriately trained, community-based assessment of patient records could assist in defining disease progression using clinical severity scores. In this study we evolved a secure, step wise process to show that pre-existing medical records may be correctly assessed by non-clinical practitioners trained to quantify disease progression. Sixty-four undergraduate students at the University of Notre Dame were expertly trained in clinical disease assessment and recognition of major and minor symptoms of NPC. Seven clinical records, randomly selected from a total of thirty seven used to establish a leading clinical severity scale, were correctly assessed to show expected characteristics of linear disease progression. Student assessment of two new records donated by NPC families to our study also revealed linear progression of disease, but both showed accelerated disease progression, relative to the current severity scale, especially at the later stages. Together, these data suggest that college students may be trained in assessment of patient records, and thus provide insight into the natural history of a disease.     

Florid Suprascapular Neuropathy after Primary Rotator Cuff Repair Attributed to Suprascapular Notch Constriction in the Setting of Double Crush Syndrome

This report describes a patient who had an open repair of a small supraspinatus tendon tear performed 6 months after an arthroscopic acromioplasty with debridement had failed to provide pain relief. Three months prior to the tendon repair, he had a two-level cervical spine discectomy and fusion (C4–5, C5–6) that improved his neck pain. Florid suprascapular neuropathy was detected 10 weeks after the open rotator cuff repair. Evidence of some nerve recovery resulted in a long period of observation. But unsatisfactory improvement warranted decompression of the suprascapular notch, which was found to be very stenotic. At surgery, there was no evidence of neuroma, cyst, or other compressing lesion or tissue. Therefore, it was ultimately hypothesized that there was an exacerbation of a preexisting, but clinically unrecognized, entrapment of the suprascapular nerve in the suprascapular notch in the setting of cervical radiculopathy (primarily C5). Retrospectively it was also concluded that had this compressive etiology been recognized, it would have favored prompt decompression rather than the long observation period. Three years was required to achieve a good result following suprascapular notch decompression. The underlying C5 radiculopathy may have created a “double crush syndrome” that contributed to the propensity for injury and the prolonged recovery. There should be heightened awareness of this problem in patients who do not have satisfactory improvement in shoulder pain from previous shoulder and neck surgery.     

Effect of suckling on cortisol, progesterone and luteinizing hormone in postpartum beef cows

Five primiparous, 3-year-old Hereford cows suckled ad libitum , were cannulated via the jugular vein and stanchioned for 2-day sampling periods, every 14 days starting 14 days after the mean calving date. On the second day of each period, calves were removed to a pen away from the cows, for 9 hours. Blood was sampled 5 min before calves were returned to their dams, as soon as possible after initiation of suckling (IOS), and at 15-min intervals for 45 min, thereafter. Cortisol, progesterone and luteinizing hormone (LH) concentrations in the serum were quantitated by radioimmunoassay. Mean serum cortisol concentrations were 7.3 +/- .7, 9.4 +/- .7, 12.1 +/- .9, 7.5 +/- .5 and 5.7 +/- .4 ng/ml (mean +/- S.E.) at -5, 0, 15, 30 and 45 min after IOS, respectively, for all cows across all periods. Cortisol concentrations, during and after suckling, tended (P<.06) to differ among sampling periods, during the postpartum interval. Serum progesterone concentrations were .28 +/- .02, .28 +/- .02, .32 +/- .05 and .24 +/- .03 ng/ml at 0, 15, 30 and 45 min after IOS, respectively, for all cows across all period, indicating that suckling had no effect on serum progesterone, and were similar at all sampling periods during the postpartum interval. Serum LH concentrations were .81 +/- .07, .77 +/- .06, .71 +/- .04, and .72 +/- .04 ng/ml at 0, 15, 30 and 45 min after IOS, respectively. During the postpartum interval, serum LH concentrations were greater (P<.01) at 71 and 85 days postpartum than at any other time.     

Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor

Diphtheria fusion proteins are chimeric proteins consisting of the catalytic and translocation domains of diphtheria toxin (DT(388)) linked through an amide bond to one of a variety of peptide ligands. The ligand targets the molecule to cells and the toxin enters the cell, inactivates protein synthesis and induces cell death. Diphtheria fusion proteins directed to human myeloid leukemic blasts are a novel class of therapeutics for patients with chemotherapy refractory myeloid leukemia. Because of the presence of interleukin-3 (IL3) receptors on myeloid leukemic progenitors and its absence from mature myeloid cells, we synthesized four bacterial expression vectors encoding DT(388) fused to human IL3. Different molecules were engineered to assess the effects of modifications on yield, purity and potency of product. The constructs differed in the size of the linker peptide between the DT(388) and IL3 domains and in the presence or absence of an oligohistidine tag on the N- or C-terminus. Escherichia coli were transformed and recombinant protein induced and purified from inclusion bodies. Similar final yields of 3-6 mg of purified protein per liter of bacterial culture were obtained with each of the four molecules. Purity ranged from 70 to 90% after partial purification by anion-exchange, size-exclusion chromatography and/or nickel affinity chromatography. Proteins were soluble and stable at 4 degrees C and -80 degrees C in phosphate-buffered saline at 0.03-0.5 mg/ml. The fusion proteins showed predicted molecular weights by SDS-PAGE, HPLC and tandem mass spectrometry and had full ADP-ribosylating activities. Each was immunoreactive with antibodies to DT(388) and IL3. Each of the fusion proteins with the exception of the one with an N-terminal oligohistidine tag showed full IL3 receptor binding affinity (K:(d) = 3 nM) and potent and selective cytotoxicity to IL3 receptor positive human myeloid leukemia cell lines (IC(50) = 5-10 pM). In contrast, the N-terminal histidine-tagged fusion protein bound IL3 receptor with a 10-fold lower affinity and was 10-fold less cytotoxic to IL3 receptor positive blasts. Thus, we report a series of novel, biologically active DT(388)IL3 fusion proteins for potential therapy of patients with receptor positive myeloid leukemias.