Biocontainment design considerations for biopharmaceutical facilities

Summary This article highlights biocontainment design considerations for biopharmaceutical manufacturing facilities. The major focus of this report is on industry's use and interpretation of the regulations with specific design recommendations for a Biosafety Level 2 — Large-Scale physical containment level as described by the National Institutes of Health Guidelines.     

A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.     

The Biosynthesis of Abscisic Acid from all-trans-{beta}-Carotene in a Cell-Free System from Citrus sinensis Exocarp

A cell-free system prepared from exocarp of Citrus sinensisfruits converted [¹⁴C]-all-trans-ß-carotene into zeaxanthin,antheraxanthin, violaxanthin and abscisic acid (ABA). ABA wasunequivocally characterized by combined capillary GC-MS. (Received August 3, 1993; Accepted August 6, 1993)     

Harleyodendron,a new genus of leguminosae (Swartzieae)

A monotypic new genus,Harleyodendron, is described from the relict coastal rainforest of eastern Brazil. Gross morphology, wood and leaf anatomy, and pollen morphology are considered.     

Stomatal responses to humidity in Opuntia inermis in relation to control of CO2 and H2O exchange patterns

Summary At constant cladode temperature the stomatal resistance of O. inermis increased when the cladode-air vapor pressure difference was increased and stomatal resistance decreased when the cladode-air vapor pressure difference was lowered. Net CO₂ fixation in the dark was very responsive to these humidity dependent changes in stomatal resistance. Net CO₂ fixation and stomatal resistance in the light did not respond to changes in cladode-air vapor pressure differences in the light under the conditions tested. When temperature response functions for dark CO₂ fixation were examined at constant ambient humidity, the reduction in dark CO₂ fixation at higher temperatures was largely due to stomatal closure in response to the increased vapor pressure difference. The water requirement for net CO₂ fixation in the dark at typical nocturnal vapor pressure differences was about 10 times lower than that of net CO₂ fixation in the light at vapor pressure differences typical of the late afternoon. The role of the stomatal responses to humidity in determining the patterns and rates of net CO₂ exchange in the light or dark, and its possible ecological significance is discussed.     

The use of species abundance estimates in marine benthic studies

The application of species abundance estimates, commonly used in terrestrial plant ecology, to marine benthic dredge data is advocated. Two possible scales were tested for their ability to measure adequately the structure of an assemblage by computing measures of evenness diversity McNaughton's dominance index, and Morisita's index of dispersion using both real counts obtained from grab samples and these counts transformed into the two respective abundance estimates. It is concluded that species abundance estimators, when applied to grab or dredge data result in significant savings in sample processing time with only a minimal loss of information about assemblage structure.     

Strong and weak immune responses across the same major histocompatibility barrier in rats

Inbred rat strains, Fischer 344 (F-344) and Lewis (LEW), share the serologicalAg-Bl allele and react very weakly in mixed lymphocyte culture (MLC). Despite this apparent identity atAg-B, these strains differ markedly in their immune responses to anAg-B disparate third strain Marshall 520 (M-520) (Ag-B6). F-344 recipients allowed M-520 heart grafts an extended survival, whereas LEW recipients rejected them rapidly. F-344 and M-520 showed a weak response in MLC in contrast to a strong response for LEW and M-520. F-344 produced antisera in response to injection of M-520 cells that had a relatively high antibody titer but low cytotoxic activity. F-344 responded to another strain, Buffalo (BUF) (alsoAg-B6), in a similar fashion. F-344 apparently can produce a strong allogeneic response, as it was able to rapidly reject heart grafts from (LEW x Brown-Norway) F₁ donors (LBN) (Ag-B 1/3). The low response of F-344 to M-520 probably was not due to shared antigens between the two strains because M-520 heart grafts underwent rapid rejection in LEW hosts highly tolerant to F-344. To explain the contrasting response of F-344 and LEW to theAg-B6 disparity, we propose that it is controlled by an immune-response gene(s); that F-344 has a low-responding allele and LEW has a high-responding allele. The data do not reveal a location for this proposed gene. The high-responding allele appears to be dominant, as M-520 hearts were rejected rapidly by (F-344 x LEW) F₁ recipients.     

Penetration of the zona-free mouse egg by capacitated epididymal sperm: Cinemicrographic observations

Conditions were established for routine cinemicrographic examination of sperm incorporation by living zona-free mouse eggs employing oil immersion objectives and Nomarski optics. Initial sperm attachment to the egg plasma membrane, which was reversible and appeared to require flagellar activity, involved localized areas of the head corresponding approximately to the position of the equatorial segment. Penetrating sperm lay flat on the egg and, during incorporation, appeared to sink into the egg cytoplasm, accompanied by short bursts of flagellar activity and subsequent rotation of the flagellum around its insertion point. Ensuing sperm head decondensation involved dissociation of individual particulate structures and a dramatic localized clearing in the egg cytoplasm. The normalcy of the penetration process and the potential applicability of this approach was attested to by the observations that polar body extrusion, male and female pronuclear formation, and migration through the egg cytoplasm in preparation for syngamy occurred in several sequences followed for extended time periods.